Quinoxalinyl-piperazinamide methods of use

ABSTRACT

The disclosed subject matter provides methods using and kits comprising a compound of formula (I) 
                         
or a pharmaceutically acceptable salt thereof.

1. PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/255,910, filed Sep. 2, 2016, and claims priority to U.S. ProvisionalApplication No. 62/214,678, filed Sep. 4, 2015; and U.S. ProvisionalApplication No. 62/289,820 filed Feb. 1, 2016, the contents of which arehereby incorporated by reference in the entirety.

2. SUMMARY OF THE INVENTION

The following summary is presented for illustrative purposes and shouldnot serve to limit the scope of the claimed subject matter.

U.S. Pat. No. 8,314,100 (issued Nov. 20, 2012), incorporated byreference herein in its entirety, discloses a compound of formula (I)

also referred to elsewhere as RX-5902, 4-(3,5-dimethoxyphenyl)-N-(7-fluoro-3-methoxyquinoxalin-2-yl)piperazine-1-carboxamide,1-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]-4-(3,5-dimethoxyphenyl)piperazine,and1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.

Other aspects of RX-5902 are described in U.S. Pat. No. 8,598,173(issued Dec. 3, 2013) and U.S. Publish Application No. 2015/0004234(published Jan. 1, 2015), both of which are incorporated by referenceherein in its entirety.

One aspect of the disclosure provides a method of treating a tumor byadministering to a subject in need thereof a solid, oral dosage formcomprising a compound of formula (I) or pharmaceutically acceptable saltthereof, wherein the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 800-15,000 hr·ng/mL after a single administration. In anembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 2,500-9,300 hr·ng/mL after a single administration. Inan embodiment, the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 2,500-9,500 hr·ng/mL after a single administration. Inan embodiment, the solid, oral dosage form may provide a C_(max) ofabout 200-1,200 ng/mL after a single administration.

Another aspect of the disclosure provides a method of treating a tumorby administering to a subject in need thereof a solid, oral dosage formcomprising a compound of formula (I) or pharmaceutically acceptable saltthereof, at a dosage of about 100-1,200 mg/day 1-7 days per week, up toabout 2,800 mg/week. In an embodiment, the dosage may be about 150-400mg/day 3-7 days per week. In an embodiment, the dosage may be about150-400 mg/day 5-7 days per week. In an embodiment, the solid, oraldosage form may be a tablet or capsule.

In any embodiment, the subject may be a human. In any embodiment, thetumor may be selected from skin, colorectal, ovarian, lung, breast,pancreatic, stomach and renal cancer. In embodiments the tumor may betriple negative (TN) breast cancer. In embodiments, the tumor may beplatinum-resistant or refractory ovarian cancer.

Another aspect of the disclosure, the method of treating a tumor,further includes administering radiation or an anti-tumor agent to thesubject. In another aspect of the disclosure, the method of treating atumor, further includes administering to the subject an anti-tumor agentselected from antimetabolites, DNA-fragmenting agents, DNA-crosslinkingagents, intercalating agents, protein synthesis inhibitors,topoisomerase I poisons, topoisomerase II poisons, microtubule-directedagents, kinase inhibitors, polyphenols, hormones, hormone antagonists,death receptor agonists, immune checkpoint inhibitors, anti-programmedcell death 1 (PD-1) receptor antibodies and anti-programmed cell deathligand 1 (PD-L1) antibodies. In an embodiment, the method comprisesadministering to the subject a PD-L1 antibody or PD-1 antibody.

Another aspect of the disclosure provides a method of treating a tumorin a subject in need thereof, including the steps of: (a) determiningwhether the subject is undergoing treatment with a CYP3A4 or CYP3A5inhibitor or inducer; and (b) if the subject is not undergoing treatmentwith a CYP3A4 or CYP3A5 inhibitor or inducer, then administering to thesubject an effective amount of a compound of formula (I) orpharmaceutically acceptable salt thereof. In embodiments, the methodfurther includes the steps of (c) monitoring the subject for an adverseevent.

Another aspect of the disclosure provides a method of inhibitingβ-catenin dependent ATPase activity of Y593 phosphorylated p68, byadministering to a subject in need thereof an effective amount of acompound of formula (I) or pharmaceutically acceptable salt thereof.

Another aspect of the disclosure provides a kit for testing potentialefficacy of a compound of formula (I) or pharmaceutically acceptablesalt thereof in treating a tumor, that includes an assay that determineswhether the tumor expresses Y593 phosphorylated p68.

Another aspect of the disclosure provides a method of treating a tumorin a subject in need thereof, by the steps of (a) collecting a sample ofthe tumor from the subject; (b) determining whether the tumor expressesY593 phosphorylated p68; and (c) if the tumor expresses the Y593phosphorylated p68, then administering to the subject an effectiveamount of a compound of formula (I) or pharmaceutically acceptable saltthereof.

Another aspect of the disclosure provides a method for preparing 4-(3,5-dimethoxyphenyl)-N-(7-fluoro-3-methoxyquinoxalin-2-yl)piperazine-1-carboxamide(RX-5902) on a commercial scale, for example, in one or more fixedreactors. In embodiments, the preparation of RX-5902 on a commercialscale can include the steps of reacting3-amino-6-fluoro-2-methoxyquinoxaline with ethyl chloroformate in anorganic solvent in the presence of a base to form a mixture; distillingthe mixture while adding ethyl acetate to form a suspension; filteringthe suspension to isolate ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl)carbonate; and reacting the ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl)carbonate with 1-(3,5-dimethoxyphenyl) piperazine hydrochloride in asecond organic solvent in the presence of a second base. In embodiments,the commercial scale production can further include the steps ofreacting 3-amino-2-chloro-6-fluoroquinoxaline with sodium methoxide inan organic solvent in the presence of a base to form a mixture; addingwater to the mixture to form a solution; cooling the solution to atemperature of about 15-20° C. to form a suspension; and filtering thesuspension to isolate 3-amino-6-fluoro-2-methoxyquinoxaline. Inembodiments, one or more of the organic solvents can be dichloromethane.In embodiments, the base can be pyridine. In embodiments, one or more ofthe distilling steps can be conducted under atmospheric pressure. Inembodiments, filtering can be by vacuum filtration. In embodiments, thesecond organic solvent can be tetrahydrofuran. In embodiments, thesecond base can be 1,8-diazabicycloundec-7-ene (DBU).

New nanoformulations providing improved oral bioavailability of RX-5902have also been discovered. The present invention is directed to new usesand methods of using the compound of formula (I) and nanoformulationsthereof. The present invention also provides an improved process toreduce impurities and significantly reduce the cost of manufacturing by,among other things, removing solvent using distillation and filtering ofthe final product. The present invention also provides newnanoformulations for improved bioavailability of RX-5902. In addition,the present invention provides dosage and exposure levels for using thecompound of formula (I) and its nanoformulations in a subject. Anotheraspect of the disclosure provides a method for preparing a mixture ofparticles of a compound of formula (1), or pharmaceutically acceptablesalt thereof, under conditions sufficient to provide a suspension. In anembodiment, the suspension may be made by a milling process. In anembodiment the milling process may be high-energy agitator milling orroller milling. In an embodiment, the milling process is high-energyagitator milling. In an embodiment, the suspension may have a D50particle size of about 200 nm or less.

Embodiments of the method can include spray drying the suspension toform a powder.

An aspect of the disclosure provides a product prepared by the method ofmaking RX-5902 by a process described herein.

3. BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph showing the plasma concentration of RX-5902 insubjects with advanced or metastatic solid tumors after a single oraladministration of RX-5902 at various doses under fasted conditions.

FIG. 2 is graph showing the mean tumor volume in mice with human renaltumor (Caki-1) xenografts following oral administration of RX-5902 at 50mg/kg and 70 mg/kg 5 days a week.

FIG. 3 is a graph showing the mean tumor volume in mice with human renaltumor (Caki-1) xenografts following oral administration of Sunitinibdaily at 60 mg/kg for 21 days and RX-5902 at 20, 40, 80 and 160 mg/kgonce a week for four weeks.

FIG. 4 is a Western blot showing the interaction of RX-5902 withMDA-MB-231 cells, in particular a band with mobility around 60 kDaprotected from protease cleavage.

FIG. 5 is a Western blot showing that the protected band of FIG. 4 wasrecognized by the antibody against p68 RNA helicase.

FIG. 6 is a Western blot confirming that p68 was phosphorylated on atyrosine residue.

FIG. 7 are graphs showing the percentage of phospho-p68 andunphosphorylated p68 bound to ³H-labeled RX-5902 in filter bindingassays. The Kd is estimated by 50% of p68 bound to RX-5902.

FIG. 8 is a bar graph showing the RNA-dependent ATPase activity of p68in the presence of 0 to 20 μM RX-5902 and total RNA extracted from 2 μgyeast.

FIG. 9 is a bar graph showing the β-catenin-dependent ATPase activity ofp68 in the presence of 0 to 20 μM RX-5902 and 1 μg β-catenin.

FIG. 10 is a graph showing the IC₅₀ determination of RX-5902 for theinhibition of β-catenin dependent ATPase activity of phospho-p68. IC₅₀was determined to be 61±7.1 nM.

FIG. 11 are Western blots showing the suppression of the expression ofcyclin D1, c-Myc, and p-c-Jun by RX-5902.

FIG. 12 is graph showing the mean tumor volume in mice with human breastcancer (MDA-MB-231) xenografts following oral administration of RX-5902at 160 mg/kg, 320 mg/kg, and 600 mg/kg once a week for three weeks,compared to Abraxane® administered intravenously at 5 mg/kg, twice aweek for three weeks.

FIG. 13 is graph showing the Kaplan-Meier survival curves in mice withhuman breast cancer (MDA-MB-231) xenografts following oraladministration of RX-5902 at 160 mg/kg, 320 mg/kg, and 600 mg/kg once aweek for three weeks, compared to Abraxane® administered intravenouslyat 5 mg/kg, twice a week for three weeks. These data are from the samestudy as shown in FIG. 12.

FIG. 14 is a graph showing Particle-Size Distribution of Agitator-MilledNanosuspension.

FIG. 15 is a DSC of Extracted RX-5902 Nanoparticles.

FIG. 16 is a Particle-Size Distribution of Spray-Dried RX-5902Nanosuspension.

FIG. 17 shows Spray-Dried RX-5902 (1000× magnification, normal light).

FIG. 18 shows Spray-Dried RX-5902 (1000× magnification, polarizedlight).

FIG. 19 shows ¹H NMR spectrum of RX-5902.

FIG. 20 shows ¹H NMR spectrum of RRT 0.975 Impurity.

FIG. 21 shows overlay of ¹H NMR spectra of RRT 0.975 Impurity (top) andRX-5902 (bottom).

FIG. 22 shows overlay of 7.0-8.0 ppm region of ¹H NMR spectra of RRT0.975 Impurity (top plot) and RX-5902 (bottom plot).

FIG. 23 shows ¹³C NMR spectrum RX-5902.

FIG. 24 shows ¹³C NMR spectrum of RRT 0.975 Impurity.

FIG. 25 shows overlay of ¹³C NMR spectra of RX-5902 (top plot) and RRT0.975 Impurity (bottom plot).

FIG. 26 shows of 108-150 ppm region of ¹³C NMR spectra of RX-5902 (topplot) and RRT 0.975 Impurity (bottom plot).

FIG. 27 shows ¹⁹F NMR spectrum of RX-5902.

FIG. 28 shows ¹⁹F NMR spectrum of RRT 0.975 Impurity.

FIG. 29 shows UV-Vis absorbance data of RX-5902 (solid line) and the RRT0.975 Impurity (dashed line).

FIG. 30 shows Liquid Chromatography-Mass Spectrometry (LC-MS) of 17.9min peak corresponding to the main RX-5902 product.

FIG. 31 shows LC-MS of 17.2 min peak corresponding to the RRT 0.975Impurity.

FIG. 32 shows RX-5902 and the RRT 0.975 Impurity having the exact[M+Na]⁺ mass of 464.

FIG. 33 is an X-Ray Powder Diffraction (XRPD) of crystalline RX-5902nanoformulation.

FIG. 34 is a detailed XRPD pattern of RX-5902 API.

FIGS. 35A and 35B are optical micrograph of the crystalline batch (left)and the crystal (right) used for the XRPD data collection.

FIG. 36 shows an XRPD pattern overlay of RX-5902 nanoformulation andRX-5902 API.

4. DETAILED DESCRIPTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. While specific exemplary embodimentsare discussed, it should be understood that this is done forillustration purposes only. A person skilled in the relevant art willrecognize that other components and configurations can be used withoutparting from the spirit and scope of the invention.

4.1 Definitions

Unless indicated otherwise, the following terms as used herein have themeanings indicated below. These meanings are intended to supplement,rather than alter, the meanings of these terms as understood in the art.

“C_(max)” refers to the maximum observed plasma concentration.

“T_(max)” refers to the time at which C_(max) is attained.

“T_(1/2) refers to the time required for the plasma concentration of adrug to reach half of its original value. “Terminal Tv2” refers to Tv2in the terminal phase.

“AUC_(0-t)” refers to the area under the plasma concentration versustime curve (AUC) from time zero to time t, wherein “t” is the lastsampling time point with measurable concentration. For example, AUC₀₋₂₄or AUC_(0-t) (0-24 hours) refers to the AUC from time zero to 24 hours,while AUC₀₋₄₈ or AUC_(0-t) (0-48 hours) refers to the AUC from time zeroto 48 hours.

“Oral dosage form” refers to a pharmaceutical composition formulated fororal administration. The oral dosage form can be formulated to provideimmediate, sustained, extended, delayed or controlled release. Examplesof an oral dosage form include tablets, capsules, granulations andgel-caps.

“Effective amount” refers to an amount of a compound or pharmaceuticalcomposition that, based on its parameters of efficacy and potential fortoxicity and the knowledge of one skilled in the art, produces a desiredeffect, such as treating or preventing a condition. An effective amountcan be administered in one or more doses.

“Contacting” refers to causing, either directly or indirectly, acompound and a cell to be in sufficient proximity as to produce adesired effect, such as inducing apoptosis or modulating protein kinase.The contacting may be performed in vitro or in vivo. For example,contacting a cell with a compound may involve delivering the compounddirectly into the cell using known techniques such as microinjection,administering the compound to a subject carrying the cell, or incubatingthe cell in a medium that includes the compound.

“Treating” refers to attaining a beneficial or desired result, such as aclinical result. In some embodiments, the beneficial or desired resultis any one or more of the following: inhibiting or suppressing the onsetor development of a condition, reducing the severity of the condition,reducing the number or severity of symptoms associated with thecondition, increasing the quality of life of a subject suffering fromthe condition, decreasing the dose of another medication required totreat the condition, enhancing the effect of another medication asubject is taking for the condition, and prolonging the survival of asubject having the condition.

“Preventing” refers to reducing the probability that a subject developsa condition which the subject does not have but is at risk ofdeveloping. “At risk” denotes that a subject has one or more riskfactors, which are measurable parameters that correlate with thedevelopment of a condition and are known in the art. A subject havingone or more of risk factors has a higher probability of developing thecondition than a subject without such risk factors.

“Subject” refers to an animal, such as a mammal, including but notlimited to, a human. Hence, the methods disclosed herein can be usefulin human therapy and veterinary applications. In one embodiment, thesubject is a mammal. In another embodiment, the subject is a human.

“Fasted” refers to a subject that has fasted from food for at least 8hours prior to treatment.

“CYP3A4 or CYP3A5 inhibitor or inducer” refers to an agent thatincreases or decreases, respectively, plasma AUC values of substratesfor the cytochrome P450 3A4 (CYP3A4) or P450 3A5 (CYP3A5) enzyme by atleast about 30 percent. Examples of a CYP3A4 or CYP3A5 inhibitor orinducer include amprenavir, aprepitant, atazanavir, barbiturate,boceprevir, bosentan, carbamazepine, chloramphenicol, ciprofloxacin,clarithromycin, cobicistat, conivaptan, darunavir, diltiazem, efavirenz,elvitegravir, erythromycin, etravirine, fluconazole, fosamprenavir,grapefruit juice, imatinib, indinavir, itraconazole, ketoconazole,lopinavir, mibefradil, modafinil, nafcillin, nefazodone, nelfinavir,phenytoin, posaconazole, rifampin, ritonavir, saquinavir, St. John'sWort, telaprevir, telithromycin, tenofovir, tipranavir, verapamil andvoriconazole.

“Adverse event” refers to any undesirable effect associated with the useof a compound or pharmaceutical composition. An adverse event may beassessed and graded according to the National Cancer Institute CommonTerminology Criteria for Adverse Events (NCI-CTCAE) version 4.03.Examples of an adverse event include febrile neutropenia, anemia,thrombocytopenia, coagulation abnormality associated with clinicalhemorrhage, diarrhea, fatigue, nausea and vomiting.

“Sensitizing” refers to increasing a cell's sensitivity to, or reducinga cell's resistance in responding to, an apoptotic signal.

“Modifying” a treatment or administration refers to reducing orincreasing the dose of an active agent, ceasing to administer the activeagent to a subject, or substituting the active agent with a differentactive agent.

“Inhibition” refers to a decrease in the expression level (such as of agene) or activity (such as of an enzyme) in the presence of an agent(such as a compound of formula (I)), relative to the expression level oractivity in the absence of that agent. The decrease can be, for example,5% or more, 10% or more, 20% or more, 25% or more, 30% or more, 40% ormore, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more,90% or more, or 95% or more. The expression level or activity can bemeasured as described herein or by techniques generally known in theart.

“Tumor cell” refers to a cell derived from a tumor.

“Tumor” refers to an abnormal growth of tissue or cells, whether benignor malignant. Examples include tumors found in prostate, lung, brain,breast, kidney, liver, lung, intestines, lymph, muscle, bone, bonemarrow, uterus, ovary, vagina, vulva, pancreas, adrenal gland, centralnervous system, peripheral nervous system, cervix, bladder, endometrium,throat, esophagus, larynx, thyroid, blood, penal, testicular, thymus,skin, spine, stomach, bile duct, small bowel, hepatobiliary tract,colorectal; colon; rectum, anus, endocrine, eye, and gall bladder.

“Cancer” refers to a malignant tumor. Cancer cells may or may not invadethe surrounding tissue and, hence, may or may not metastasize to newbody sites. Cancer encompasses carcinomas, which are cancers ofepithelial cells; carcinomas include squamous cell carcinomas,adenocarcinomas, melanomas, and hepatomas. Cancer also encompassessarcomas, which are tumors of mesenchymal origin; sarcomas includeosteogenic sarcomas, leukemias, and lymphomas. Cancers may involve oneor more neoplastic cell type.

“Anti-tumor agent” refers to any agent useful for treating or preventingtumor. Examples of an anti-tumor agent include the active agentsdescribed in Pharmaceutical Compositions, infra. In embodiments, theanti-tumor agent in addition to RX-5902 is selected fromantimetabolites, DNA-fragmenting agents, DNA-crosslinking agents,intercalating agents, protein synthesis inhibitors, topoisomerase Ipoisons, topoisomerase II poisons, microtubule directed agents, kinaseinhibitors, polyphenols, hormones, hormone antagonists, death receptoragonists, immune checkpoint inhibitors, anti-programmed cell death 1(PD-1) receptor antibodies and anti-programmed cell death ligand 1(PD-L1) antibodies. In other embodiments, the additional anti-tumoragent is a PD-1 receptor antibody. In other embodiments, the additionalanti-tumor agent is pembrolizumab. In other embodiments, the additionalanti-tumor agent is nivolumab. In other embodiments, the additionalanti-tumor agent is duryalumab. In other embodiments, the additionalanti-tumor agent is combination of nivolumab and pembrolizumab.

“Radiation” refers to any radiation useful for treating or preventingtumor. Examples of radiation include X-rays, gamma rays, and chargedparticles. The radiation may be delivered by any form of radiationtherapy, such as external beam radiotherapy (EBRT, XBRT or teletherapy),brachytherapy (internal radiation therapy or sealed source therapy),intraoperative radiotherapy, or systemic radiation therapy.

“Y593 phosphorylated p68” or “Y593 phospho-p68” refers to the p68 RNAhelicase (DDX5) phosphorylated at the tyrosine 593 residue.

“p68 RNA helicase,” also known as “ATP-dependent RNA helicase DDX5” or“DEAD box protein 5,” refers to an enzyme that in humans is encoded bythe DDX5 gene.

“Commercial scale” refers to the preparation of a product in a quantitythat would be suitable for its manufacture for sale and distribution tothe public. Commercial scale is distinguished from laboratory or benchscale in which the quantity produced is suitable for research purposes.Commercial scale is also distinguished from laboratory or bench scalesynthesis in using reagents and methods that minimize use or waste ofhazardous substances to minimize disposal and clean-up costs. In certainembodiments, the commercial scale methods disclose herein yield a singlebatch quantity of at least about 0.5 kg, 1.0 kg or 1.5 kg.

“Reacting” refers to combining two or more reagents under appropriateconditions (e.g., temperature, pressure, pH, concentration) to produce adesired product. The desired product may not necessarily result directlyfrom the combination of the two or more reagents; i.e., one or moreintermediates ay be produced which ultimately lead to the formation ofthe desired product.

“Distillation” or “distilling” refers to separating compounds based ontheir different volatilities, such as by vaporization or subsequentcondensation.

“Filtration” or “filtering” refers to separating solids from a liquid,such as by vacuum, gravity or pressure. “Vacuum filtration” refers to atechnique for extracting solids from a liquid mixture, in which vacuumsuction is applied to draw the mixture through a filter, such as filterpaper in a Büchner funnel.

“Atmospheric pressure” refers to open air pressure, as opposed to thepressure in a vacuum or enclosed chamber. Atmospheric pressure istypically about 760 Torr, but it can vary depending inter alia on theevaluation of the manufacturing facility.

“Vacuum filtration” refers to a technique for extracting solids from aliquid mixture, in which vacuum suction is applied to draw the mixturethrough a filter, such as filter paper in a Büchner funnel.

“Fixed reactor” refers to a reactor that is fixed in place and does notmove.

“Particle size” refers to the particle dimension of an activepharmaceutical ingredient (API), such as the compound of formula (I) orpharmaceutically acceptable salt thereof, as determined by any particlesize measuring technique known in the art. Non limiting examples of suchtechnique include laser diffraction, dynamic light scattering and imageanalysis performed, for example, using an analyzer, such as oneavailable from Malvern, Sympatec, Microtac or Horiba.

“D10” refers to the particle size at 10% in the cumulative distribution,meaning that 10% of the particles have a particle size of less than D10,and 90% of the particles have a particle size of more than D10. “D50”refers to the median particle size or the particle size at 50% in thecumulative distribution, meaning that 50% of the particles have aparticle size of less than D50, and 50% of the particles have a particlesize of more than D50. “D90” refers to the median particle size or theparticle size at 90% in the cumulative distribution, meaning that 90% ofthe particles have a particle size of less than D90, and 10% of theparticles have a particle size of more than D90. The cumulativedistribution may be based on the volume, mas, number or surface area ofthe particles. Unless otherwise specified, the cumulative distributionis based on the volume of the particles.

“Lyophilizing” refers to using a freeze-drying process to removesubstantially one or more solvents from a product by freezing theproduct and then reducing the surrounding pressure to allow the frozensolvent(s) in the product to sublimate directly from the solid phase tothe gas phase.

“Spray drying” refers to a method of producing a dry powder from aliquid or slurry by rapidly drying with a hot gas.

“Such as” has the same meaning as “such as but not limited to.”Similarly, “include” has the same meaning as “include but not limitedto,” while “including” has the same meaning as “including but notlimited to.”

The singular forms “a.” “or,” and “the” include plural referents unlessthe context dictates otherwise. Thus, for example, a reference to “acompound” may include one or more compound(s) and/or equivalent(s)thereof.

Any numerical range disclosed herein encompasses the and lower limitsand each intervening value, unless otherwise specified.

Other than in the working examples, or where otherwise indicated,numerical values (such as numbers expressing quantities of ingredients,reaction conditions) as used in the specification and claims aremodified by the term “about”. Accordingly, unless indicated to thecontrary, such numbers are approximations that may vary depending uponthe desired properties sought to be obtained. At the very least, and notas an attempt to limit the application of the doctrine of equivalents tothe scope of the claims, each numerical parameter should be construed inlight of the number of significant digits and ordinary roundingtechniques.

While the numerical parameters setting forth the scope of the disclosedsubject matter are approximations, the numerical values set forth in theworking examples are reported as precisely as possible. Any numericalvalue, however, inherently contains certain errors necessarily resultingfrom the standard deviation found in its respective testingmeasurements.

Unless defined otherwise, the meanings of technical and scientific termsas used herein are those commonly understood by one of ordinary skill inthe art to which the disclosed subject matter belongs.

4.2 Methods of Treating or Preventing a Tumor

One aspect of the disclosure provides a method of treating or preventinga tumor, by administering to a subject in need thereof an effectiveamount of a compound of formula (I) or pharmaceutically acceptable saltthereof, after the subject has fasted from food for at least about 8hours. In another embodiment, the subject continues to fast from foodfor at least about 3 hours after administration.

Another aspect of the disclosure provides a method of treating orpreventing a tumor, by administering to a subject in need thereof asolid, oral dosage form including a compound of formula (I) orpharmaceutically acceptable salt thereof, wherein the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 800-15,000 hr·ng/mLafter a single administration.

Another aspect of the disclosure provides a method of treating orpreventing a tumor, comprising administering to a subject in needthereof a solid, oral dosage form including a compound of formula (I) orpharmaceutically acceptable salt thereof, wherein the solid, oral dosageform provides an AUC_(0-t) (0-168 hours; i.e., one week) of about1,000-70,000 hr·ng/mL after one week, 1-7 days per week, ofadministration.

Another aspect of the disclosure provides a method of treating orpreventing a tumor, by administering to a subject in need thereof asolid, oral dosage form including a compound of formula (I) orpharmaceutically acceptable salt thereof, at a dosage of about 100-1,200mg/day 1-7 days per week. In embodiments, the compound of formula (I) orpharmaceutically acceptable salt thereof may be administered in anamount of up to about 3,000 mg/week. In embodiments, the compound offormula (I) or pharmaceutically acceptable salt thereof may beadministered in an amount of up to about 2,800 mg/week. In oneembodiment, the dosage is about 100-500 mg/day 3-7 days per week. Inanother embodiment, the dosage is about 100-500 mg/day 5-7 days perweek. In another embodiment, the dosage is about 150-400 mg/day 3-7 daysper week. In another embodiment, the dosage is about 150-400 mg/day 5-7days a week.

Another aspect of the disclosure provides a method of treating orpreventing a tumor in a subject in need thereof, by: (a) determiningwhether the subject is undergoing treatment with a CYP3A4 or CYP3A5inhibitor or inducer; and (b) if the subject is not undergoing treatmentwith a CYP3A4 or CYP3A5 inhibitor or inducer, then administering to thesubject an effective amount of a compound of formula (I) orpharmaceutically acceptable salt thereof. In one embodiment, the subjectis undergoing treatment with a CYP3A4 or CYP3A5 inhibitor. In anotherembodiment, the subject is undergoing treatment with a CYP3A4 or CYP3A5inducer.

Another aspect of the disclosure provides a method of treating orpreventing a tumor in a subject in need thereof, by: (a) determiningwhether the subject is undergoing treatment with a CYP3A4 or CYP3A5inhibitor or inducer; (b) if the subject is undergoing treatment with aCYP3A4 or CYP3A5 inhibitor or inducer, then administering to the subjectan effective amount of a compound of formula (I) or pharmaceuticallyacceptable salt thereof; and (c) monitoring the subject for an adverseevent. In another embodiment, the method further comprises modifying thetreatment with CYP3A4 or CYP3A5 inhibitor or inducer or theadministration of the compound of formula (I) or pharmaceuticallyacceptable salt thereof if an adverse event is detected. In oneembodiment, the subject is undergoing treatment with a CYP3A4 or CYP3A5inhibitor. In another embodiment, the subject is undergoing treatmentwith a CYP3A4 or CYP3A5 inducer.

Modifying treatment may include, for example, reducing or increasing thedose of the CYP3A4 or CYP3A5 inhibitor or inducer, or reducing orincreasing the dose of RX-5902. In some embodiments, modifying includesone or more of the following: reducing the dose of the CYP3A4 or CYP3A5inhibitor, increasing the dose of the CYP3A4 or CYP3A5 inducer,increasing the dose of RX-5902 if the subject is undergoing treatmentwith a CYP3A4 or CYP3A5 inhibitor, and decreasing the dosage of RX-5902if the subject is undergoing treatment with a CYP3A4 or CYP3A5 inducer.

In one embodiment, the CYP3A4 or CYP3A5 inhibitor or inducer is abarbiturate, bosentan, carbamazepine, efavirenz, etravirine, modafinil,nafcillin, phenytoin, rifampin, St. John's Wort, glucocorticoid,nevirapine, oxcarbazepine, phenobarbital, pioglitazone, rifabutin,troglitazone and grapefruit juice. In another embodiment, the CYP3A4 orCYP3A5 inhibitor or inducer is grapefruit juice. In another embodiment,the CYP3A4 or CYP3A5 inhibitor or inducer is St. John's Wort.

Another aspect of the disclosure provides a method of treating a tumorin a subject in need thereof by the steps of:

(a) collecting a sample of the tumor from the subject;

(b) determining whether the tumor expresses Y593 phosphorylated p68; and

(c) if the tumor expresses the Y593 phosphorylated p68, thenadministering to the subject an effective amount of a compound offormula (I) or pharmaceutically acceptable salt thereof.

Additional embodiments of the methods disclosed herein are describedbelow.

In one embodiment, the compound of formula (I) or pharmaceuticallyacceptable salt thereof is administered 5-7 days per week. In anotherembodiment, the compound of formula (I) or pharmaceutically acceptablesalt thereof is administered 5-7 days per week for 4 consecutive weeksor for 3 consecutive weeks followed by 1 off-week during which thecompound of formula (I) or pharmaceutically acceptable salt thereof isnot administered. In another embodiment, the compound of formula (I) orpharmaceutically acceptable salt thereof is administered for up to 12dosing cycles, wherein each dosing cycle consists of either 3consecutive weeks of treatment followed by 1 off-week, or 4 consecutiveweeks of treatment.

In another embodiment, the compound of formula (I) or pharmaceuticallyacceptable salt thereof is formulated as a solid, oral dosage form. Inanother embodiment, the solid, oral dosage form is a tablet. In anotherembodiment, the solid, oral dosage form is a capsule. In anotherembodiment, the oral, solid dosage form is a tablet or capsulecomprising nanoparticles of the compound of formula (I) orpharmaceutically acceptable salt thereof.

In another embodiment, the solid, oral dosage form, compound of formula(I) or pharmaceutically acceptable salt thereof is administered afterthe subject has fasted from food for at least about 8 hours. In anotherembodiment, the subject fasts from food for at least about 3 hours afteradministration. In another embodiment, the solid, oral dosage form,compound of formula (I) or pharmaceutically acceptable salt thereof isadministered with food.

In another embodiment, the solid, oral dosage form provides a T_(max) ofabout 1-6 hours after a single administration. In another embodiment,the solid, oral dosage form provides a T_(max) of about 2-6 hours aftera single administration. In another embodiment, the solid, oral dosageform provides a T_(max) of about 2 hours after a single administration.In another embodiment, the solid, oral dosage form provides a T_(max) ofabout 3 hours after a single administration. In another embodiment, thesolid, oral dosage form provides a T_(max) of about 4 hours after asingle administration. In another embodiment, the solid, oral dosageform provides a T_(max) of about 5 hours after a single administration.In another embodiment, the solid, oral dosage form provides a T_(max) ofabout 6 hours after a single administration.

In another embodiment, the solid, oral dosage form provides a C_(max) ofabout 90-1,200 ng/mL after a single administration. In an embodiment,the solid, oral dosage form may provide a C_(max) of about 200-1200ng/mL after a single administration. In another embodiment, the solid,oral dosage form provides a C_(max) of about 200-800 ng/mL after asingle administration. In another embodiment, the solid, oral dosageform provides a C_(max) of about 300-700 ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides a C_(max) of about 200-300 ng/mL after a single administration.In another embodiment, the solid, oral dosage form provides a C_(max) ofabout 300-400 ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides a C_(max) of about400-500 ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides a C_(max) of about 500-600 ng/mL aftera single administration. In another embodiment, the solid, oral dosageform provides a C_(max) of about 600-700 ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides a C_(max) of about 700-800 ng/mL after a single administration.

In another embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-24 hours) of about 800-15,000 hr·ng/mL after a single administration.In another embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-24 hours) of about 2,000-15,000 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-24 hours) of about 2,000-8,500 hr·ng/mL after asingle administration. In another embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 2,000-10,000 hr·ng/mLafter a single administration. In an embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 2,500-9,500 hr·ng/mLafter a single administration. In an embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 2,500-9,300 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 3,000-7,500hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides an AUC_(0-t) (0-24 hours) of about3,500-7,000 hr·ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 3,000-5,000 hr·ng/mL after a single administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-24 hours) of about 4,000-6,500 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-24 hours) of about 4,500-6,000 hr·ng/mL after asingle administration. In another embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 2,000-3,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 3,000-4,000hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides an AUC_(0-t) (0-24 hours) of about4,000-5,000 hr·ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 5,000-6,000 hr·ng/mL after a single administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-24 hours) of about 6,000-7,000 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-24 hours) of about 7,000-8,000 hr·ng/mL after asingle administration. In another embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 8,000-9,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 9,000-10,000hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides an AUC_(0-t) (0-24 hours) of about10,000-11,000 hr·ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-24hours) of about 11,000-12,000 hr·ng/mL after a single administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-24 hours) of about 12,000-13,000 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-24 hours) of about 13,000-14,000 hr·ng/mL aftera single administration. In another embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-24 hours) of about 15,000-16,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 4,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 4,500 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 5,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 5,500 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 6,000 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 6,500 hr·ng/mLafter a single administration. In another embodiment, the solid, oraldosage form provides a C_(max) of about 90-1100 ng/mL and an AUC_(0-t)(0-24 hours) of about 800-15,000 hr·ng/mL after a single administration.In another embodiment, the solid, oral dosage form provides a C_(max) ofabout 200-1,200 ng/mL and an AUC_(0-t) (0-24 hours) of about 2,500-9,500hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides a C_(max) of about 200-1,200 ng/mL andan AUC_(0-t) (0-24 hours) of about 2,500-9,300 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides a C_(max) of about 200-800 ng/mL and an AUC_(0-t) (0-24 hours)of about 2,000-15,000 hr·ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides a C_(max) of about200-300 ng/mL and an AUC_(0-t) (0-24 hours) of about 2,000-4,000hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides a C_(max) of about 300-400 ng/mL and anAUC_(0-t) (0-24 hours) of about 4,000-7,000 hr·ng/mL after a singleadministration. In another embodiment, the solid, oral dosage formprovides a C_(max) of about 400-500 ng/mL and an AUC_(0-t) (0-24 hours)of about 5,000-6,000 hr·ng/mL after a single administration. In anotherembodiment, the solid, oral dosage form provides a C_(max) of about600-700 ng/mL and an AUC_(0-t) (0-24 hours) of about 14,000-15,000hr·ng/mL after a single administration. In another embodiment, thesolid, oral dosage form provides a C_(max) of about 700-800 ng/mL and anAUC_(0-t) (0-24 hours) of about 10,000-11,000 hr·ng/mL after a singleadministration.

In another embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-168 hours) of about 1,000-70,000 hr·ng/mL after one week, 1-7 daysper week, of administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-168 hours) of about 10,000-70,000hr·ng/mL after one week, 3-7 days per week, of administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-168 hours) of about 20,000-60,000 hr·ng/mL after one week, 3-7 daysper week, of administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-168 hours) of about 20,000-70,000hr·ng/mL after one week, 5-7 days per week, of administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-168 hours) of about 30,000-60,000 hr·ng/mL after one week, 5-7 daysper week, of administration. In another embodiment, the solid, oraldosage form provides an AUC_(0-t) (0-168 hours) of about 4,000-10,000hr·ng/mL after one week, one day per week, of administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-168hours) of about 6,000-8,000 hr·ng/mL after one week, one day per week,of administration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-168 hours) of about 6,500-7,500 hr·ng/mL afterone week, one day per week, of administration. In another embodiment,the solid, oral dosage form provides an AUC_(0-t) (0-168 hours) of about7,000 hr·ng/mL after one week, one day per week, of administration. Inanother embodiment, the solid, oral dosage form provides an AUC_(0-t)(0-168 hours) of about 10,000-35,000 hr·ng/mL after one week, 3 days perweek, of administration. In another embodiment, the solid, oral dosageform provides an AUC_(0-t) (0-168 hours) of about 15,000-30,000 hr·ng/mLafter one week, 3 days per week, of administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-168hours) of about 20,000-25,000 hr·ng/mL after one week, 3 days per week,of administration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-168 hours) of about 20,000-60,000 hr·ng/mLafter one week, 5 days per week, of administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-168hours) of about 25,000-55,000 hr·ng/mL after one week, 5 days per week,of administration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-168 hours) of about 30,000-50,000 hr·ng/mLafter one week, 5 days per week, of administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-168hours) of about 30,000-75,000 hr·ng/mL after one week, 7 days per week,of administration. In another embodiment, the solid, oral dosage formprovides an AUC_(0-t) (0-168 hours) of about 35,000-70,000 hr·ng/mLafter one week, 7 days per week, of administration. In anotherembodiment, the solid, oral dosage form provides an AUC_(0-t) (0-168hours) of about 40,000-65,000 hr·ng/mL after one week, 7 days per week,of administration.

In another embodiment, the compound of formula (I) or pharmaceuticallyacceptable salt thereof is administered at a dosage of about 100-1,200mg/day 1-7 days per week, up to about 3,000 mg/week. In anotherembodiment, the dosage is about 100-1,200 mg/day 1-7 days per week, upto about 2,800 mg/week. In another embodiment, the dosage is about100-1,200 mg/day 1-7 days per week, up to about 2,000 mg/week. Inanother embodiment, the dosage is about 100-600 mg/day 1-7 days perweek. In another embodiment, the dosage is about 100-600 mg/day 3-7 daysper week. In another embodiment, the dosage is about 100-600 mg/day 5-7days per week. In another embodiment, the dosage is about 100-600 mg/day3 days per week. In another embodiment, the dosage is about 100-600mg/day 4 days per week. In another embodiment, the dosage is about100-600 mg/day 5 days per week. In another embodiment, the dosage isabout 100-600 mg/day 6 days per week. In another embodiment, the dosageis about 100-600 mg/day 7 days per week.

In another embodiment, the dosage is about 200-500 mg/day 1-7 days perweek. In another embodiment, the dosage is about 200-500 mg/day 3-7 daysper week. In another embodiment, the dosage is about 200-500 mg/day 5-7days per week. In another embodiment, the dosage is about 200-500 mg/day3 days per week. In another embodiment, the dosage is about 200-500mg/day 4 days per week. In another embodiment, the dosage is about200-500 mg/day 5 days per week. In another embodiment, the dosage isabout 200-500 mg/day 6 days per week. In another embodiment, the dosageis about 200-500 mg/day 7 days per week.

In another embodiment, the dosage is about 150-400 mg/day 1-7 days perweek. In another embodiment, the dosage is about 150-400 mg/day 3-7 daysper week. In another embodiment, the dosage is about 150-400 mg/day 5-7days per week. In another embodiment, the dosage is about 150-400 mg/day3 days per week. In another embodiment, the dosage is about 150-400mg/day 4 days per week. In another embodiment, the dosage is about150-400 mg/day 5 days per week. In another embodiment, the dosage isabout 150-400 mg/day 6 days per week. In another embodiment, the dosageis about 150-400 mg/day 7 days per week.

In another embodiment, the dosage is about 200 mg/day 1-7 days per week.In another embodiment, the dosage is about 200 mg/day 3-7 days per week.In another embodiment, the dosage is about 200 mg/day 5-7 days per week.In another embodiment, the dosage is about 200 mg/day 3 days per week.In another embodiment, the dosage is about 200 mg/day 4 days per week.In another embodiment, the dosage is about 200 mg/day 5 days per week.In another embodiment, the dosage is about 200 mg/day 6 days per week.In another embodiment, the dosage is about 200 mg/day 7 days per week.

In another embodiment, the dosage is about 250 mg/day 1-7 days per week.In another embodiment, the dosage is about 250 mg/day 3-7 days per week.In another embodiment, the dosage is about 250 mg/day 5-7 days per week.In another embodiment, the dosage is about 250 mg/day 3 days per week.In another embodiment, the dosage is about 250 mg/day 4 days per week.In another embodiment, the dosage is about 250 mg/day 5 days per week.In another embodiment, the dosage is about 250 mg/day 6 days per week.In another embodiment, the dosage is about 250 mg/day 7 days per week.

In another embodiment, the dosage is about 300 mg/day 1-7 days per week.In another embodiment, the dosage is about 300 mg/day 3-7 days per week.In another embodiment, the dosage is about 300 mg/day 5-7 days per week.In another embodiment, the dosage is about 300 mg/day 3 days per week.In another embodiment, the dosage is about 300 mg/day 4 days per week.In another embodiment, the dosage is about 300 mg/day 5 days per week.In another embodiment, the dosage is about 300 mg/day 6 days per week.In another embodiment, the dosage is about 300 mg/day 7 days per week.

In another embodiment, the dosage is about 350 mg/day 1-7 days per week.In another embodiment, the dosage is about 350 mg/day 3-7 days per week.In another embodiment, the dosage is about 350 mg/day 5-7 days per week.In another embodiment, the dosage is about 350 mg/day 3 days per week.In another embodiment, the dosage is about 350 mg/day 4 days per week.In another embodiment, the dosage is about 350 mg/day 5 days per week.In another embodiment, the dosage is about 350 mg/day 6 days per week.In another embodiment, the dosage is about 350 mg/day 7 days per week.

In another embodiment, the dosage is about 400 mg/day 1-7 days per week.In another embodiment, the dosage is about 400 mg/day 3-7 days per week.In another embodiment, the dosage is about 400 mg/day 5-7 days per week.In another embodiment, the dosage is about 400 mg/day 3 days per week.In another embodiment, the dosage is about 400 mg/day 4 days per week.In another embodiment, the dosage is about 400 mg/day 5 days per week.In another embodiment, the dosage is about 400 mg/day 6 days per week.In another embodiment, the dosage is about 400 mg/day 7 days per week.

Daily dosage is based upon an adult human having a weight or body massof about 60-80 kg. Thus, for a range of about 100-1,200 mg/day, thedosage can range from about 1-20 mg/kg/day up to about 50 mg/kg/week.Additional dosages based on subject weight may be readily calculatedfrom these values. Similarly, persons skilled in the art will be able tocalculate dosages for other species based on known correlations to humandosages.

The total daily dose can be administered in one or more doses. In oneembodiment, the oral dosage form is administered once daily. In anotherembodiment, the oral dosage form is administered twice daily. In anotherembodiment, the oral dosage form is administered three times daily. Inanother embodiment, the oral dosage form is administered four timesdaily.

In embodiments, the oral dosage form is administered at a dosage of upto about 12,000 mg/month. The total monthly dose can be administered 1-7days per week either for three weeks followed by one week of rest, orfor four weeks without rest. For each week of treatment, the oral dosageform may be administered 1-7 days per week. In one embodiment, the oraldosage form is administered for three weeks followed by one week ofrest. In another embodiment, the oral dosage form is administered 3-7days per week for three weeks followed by one week of rest. In anotherembodiment, the oral dosage form is administered 5-7 days per week forthree weeks followed by one week of rest. In another embodiment, theoral dosage form is administered daily for three weeks followed by oneweek of rest. In another embodiment, the oral dosage form isadministered daily for 28 days. Each dosing cycle consists of either 3weeks of treatment followed by 1 week of rest, or 4 continuous weeks oftreatment. The dosing cycle may be repeated as often as necessary asdetermined by a person skilled in the art. In one embodiment, the oraldosage form is administered for up to 12 dosing cycles. In oneembodiment, the oral dosage form is administered for up to 6 dosingcycles.

In any embodiment, the tumor is selected from gastrointestinal,genitourinary, skin, colorectal (colon or rectal), ovarian, lung,breast, pancreatic, stomach and renal cancer. In another embodiment, thetumor is gastrointestinal cancer. In some embodiments, the tumor isgenitourinary cancer. In another embodiment, the tumor is skin cancer.In another embodiment, the tumor is melanoma. In another embodiment, thetumor is colorectal cancer. In another embodiment, the tumor is coloncancer. In another embodiment, the tumor is rectal cancer. In anotherembodiment, the tumor is K-Ras mutant colon cancer. In anotherembodiment, the tumor is ovarian cancer. In another embodiment, thetumor is platinum-resistant or -refractory (e.g., cisplatin- orcarboplatin-resistant) ovarian cancer. In another embodiment, the tumoris lung cancer. In another embodiment, the tumor is non-small cell lungcancer. In another embodiment, the tumor is breast cancer. In anotherembodiment, the tumor is triple-negative (TN) breast cancer. In anotherembodiment, the tumor is metastatic breast cancer. In anotherembodiment, the tumor is pancreatic cancer. In another embodiment, thetumor is stomach cancer. In another embodiment, the tumor is renalcancer.

In another embodiment, the subject is a mammal. In another embodiment,the subject is a human.

4.3 Methods of Inhibiting β-Catenin Dependent ATPase Activity of Y593Phosphorylated p68

Another aspect of the disclosure provides a method of inhibitingβ-catenin dependent ATPase activity of Y593 phosphorylated p68,comprising administering to a subject in need thereof an effectiveamount of a compound of formula (I) or pharmaceutically acceptable saltthereof.

4.4 Methods of Predicting Efficacy of Treatment

Another aspect of the disclosure provides a method of predictingefficacy of treatment of a subject in need thereof with a compound offormula (I) or pharmaceutically acceptable salt thereof, comprising:

(a) collecting a sample of the tumor from the subject;

(b) determining whether the tumor expresses Y593 phosphorylated p68.

In one embodiment, the method also includes administering the compoundof formula (I) or pharmaceutically acceptable thereof to the subject ifthe tumor expresses Y593 phosphorylated p68.

In another embodiment, the method also includes determining whether thecompound of formula (I) or pharmaceutically acceptable salt thereofinhibits β-catenin dependent ATPase activity of the Y593 phosphorylatedp68. In another embodiment, the method includes administering thecompound of formula (I) or pharmaceutically acceptable thereof to thesubject if inhibition of β-catenin dependent ATPase activity andexpression of Y593 phosphorylated p68 are detected.

In another embodiment, the method also includes the step of determiningwhether the compound of formula (I) or pharmaceutically acceptablethereof inhibits RNA-dependent ATPase activity of the Y593phosphorylated p68. In another embodiment, the method includesadministering the compound of formula (I) or pharmaceutically acceptablesalt thereof to the subject only if inhibition of RNA-dependent ATPaseactivity is not detected.

In another embodiment, method also includes determining whether thecompound of formula (I) or pharmaceutically acceptable salt thereofinhibits translocation of β-catenin into the tumor's cell nucleus. Inanother embodiment, the method includes determining whether the compoundof formula (I) or pharmaceutically acceptable salt thereof decreasesintracellular levels of β-catenin. In another embodiment, the methodincludes determining whether the compound of formula (I) orpharmaceutically acceptable salt thereof inhibits expression of one ormore genes regulated by β-catenin. In another embodiment, the methodalso includes administering the compound of formula (I) orpharmaceutically acceptable salt thereof to the subject only ifinhibition of the expression of the one or more genes regulated byβ-catenin is detected. In another embodiment, the method also includesadministering the compound of formula (I) or pharmaceutically acceptablesalt thereof to the subject only if inhibition of the β-catenin-TCF-4mediated transcription activity is detected. In another embodiment, themethod also includes administering the compound of formula (I) orpharmaceutically acceptable salt thereof to the subject only ifinhibition of the Wnt signaling activity is detected. In anotherembodiment, the one or more genes are selected from cyclin D1, c-Myc,Axing, Surviv1, and p-c-Jun.

In embodiments, the subject is a mammal. In another embodiment, thesubject is a human.

In any embodiment, the tumor is selected from gastrointestinal,genitourinary, skin, colorectal (colon or rectal), ovarian, lung,breast, pancreatic, stomach and renal cancer. In another embodiment, thetumor is gastrointestinal cancer. In some embodiments, the tumor isgenitourinary cancer. In another embodiment, the tumor is skin cancer.In another embodiment, the tumor is melanoma. In another embodiment, thetumor is colorectal cancer. In another embodiment, the tumor is coloncancer. In another embodiment, the tumor is rectal cancer. In anotherembodiment, the tumor is K-Ras mutant colon cancer. In anotherembodiment, the tumor is ovarian cancer. In another embodiment, thetumor is platinum-resistant or -refractory (e.g., cisplatin- orcarboplatin-resistant) ovarian cancer. In another embodiment, the tumoris lung cancer. In another embodiment, the tumor is non-small cell lungcancer. In another embodiment, the tumor is breast cancer. In anotherembodiment, the tumor is triple-negative (TN) breast cancer. In anotherembodiment, the tumor is metastatic breast cancer. In anotherembodiment, the tumor is pancreatic cancer. In another embodiment, thetumor is stomach cancer. In another embodiment, the tumor is renalcancer.

4.5 Kits for Testing Efficacy of Treatment

Another aspect of the disclosure provides a kit for testing potentialefficacy of a compound of formula (I) or pharmaceutically acceptablesalt thereof in treating a tumor, where the kit includes an assay thatdetermines whether the tumor expresses Y593 phosphorylated p68.

In one embodiment, the kit also includes an assay that detectsinhibition of β-catenin dependent ATPase activity of the Y593phosphorylated p68. In another embodiment, the kit further includes anassay that detects intracellular levels (e.g., cytosolic and nuclearlevels) of β-catenin. In another embodiment, the kit includes an assaythat detects inhibition of the expression of one or more genes regulatedby β-catenin. In another embodiment, the one or more genes are selectedfrom cyclin D1, c-Myc and p-c-Jun.

4.6 Pharmaceutical Compositions

In any of the methods and kits provided herein, the compound of formula(I) or pharmaceutically acceptable salt thereof may be in apharmaceutical composition. Such pharmaceutical composition can beprepared as any appropriate unit dosage form. For example, thepharmaceutical compositions can be formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, as drenches, tablets (such as thosetargeted for buccal, sublingual and systemic absorption, includingover-encapsulation tablets), capsules (such as hard, soft, dry-filled,liquid-filled, gelatin, non-gelatin or over-encapsulation capsules),caplets, boluses, powders, sachets, granules, pastes, mouth sprays,troches, lozenges, pellets, syrups, suspensions, elixirs, liquids,liposomes, emulsions and microemulsions; or (2) parenteraladministration by, for example, subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension.Additionally, the pharmaceutical compositions can be formulated forimmediate, sustained, extended, delayed or controlled release.

In one embodiment, the pharmaceutical composition is formulated for oraladministration. In another embodiment, the pharmaceutical composition isa solid, oral dosage form. In another embodiment, the pharmaceuticalcomposition is a solid, oral dosage form that provides a T_(max),C_(max), AUC_(0-t) or combination thereof as described herein (seeSection 4.2). In another embodiment, the pharmaceutical composition is atablet or capsule. In another embodiment, the pharmaceutical compositionis a tablet. In another embodiment, the pharmaceutical composition is acapsule. In another embodiment, the tablet or capsule is formulated forimmediate release. In another embodiment, the tablet or capsule isformulated for sustained, extended, delayed or controlled release.

In another embodiment, the tablet or capsule also include at least onepharmaceutically acceptable carrier. Carriers include any substance thatmay be administered with the pharmaceutical composition with theintended purpose of facilitating, assisting, or helping theadministration or other delivery of the pharmaceutical compositionand/or improve the bioavailability of the pharmaceutical composition.Carriers may include any liquid, solid, semisolid, gel aerosol or otherssubstances that may be combined with the pharmaceutical composition toaid in its administration. Such carriers may further include binderssuch as ethyl cellulose, carboxymethylcellulose, microcrystallinecellulose, or gelatin; excipients such as starch, lactose or dextrins;disintegrating agents such as alginic acid, sodium alginate, Primogel,and corn starch; lubricants such as magnesium stearate or Sterotex;glidants such as colloidal silicon dioxide; sweetening agents such assucrose or saccharin, a flavoring agent such as peppermint, methylsalicylate or orange flavoring, or coloring agents. Further examples ofcarriers may include polyethylene glycol, cyclodextrin, oils, or anyother similar liquid carrier that may be formulated into a capsule.Examples of suitable carriers may include diluents, adjuvants,excipients, water, lipidic formulations and oils, such as petroleum,animal, vegetable or synthetic oils. Suitable excipients may includewater-insoluble surfactants, water-soluble surfactants, and hydrophiliccosolvents. Suitable oils may include tri, di or monoglycerides.

In another embodiment, nanoparticles of RX-5902 can be prepared andformulated as suspensions, tablets, capsules or other dosage forms, suchas disclosed in U.S. Pat. Pub. No. US20150004234 (published Jan. 1,2015). In one embodiment, the nanoparticles have a median particle size(D50) of less than about 1,000 nm. In another embodiment, thenanoparticles have a median particle size (D50) of less than about 500nm. In another embodiment, nanoparticles of RX-5902 are formulated as asuspension. In another embodiment, the suspension is dried, such as bylyophilization, to form a powder. In another embodiment, the powder iscombined with one or more pharmaceutically acceptable excipients. Inanother embodiment, the powder is encapsulated into capsules.

The composition and preparation of capsules are well known in the art.For example, capsules may be prepared from gelatin (e.g., Type A, TypeB), carrageenan (e.g., kappa, iota, lambda) and/or modified cellulose(e.g., hydroxypropyl methyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose acetate succinate, hydroxypropyl methyl cellulosephthalate, cellulose acetate phthalate), and optionally one or moreexcipients such as oils (e.g., fish oil, olive oil, corn oil, soybeanoil, coconut oil, tri-, di- and monoglycerides), plasticizers (e.g.,glycerol, glycerin, sorbitol, polyethylene glycol, citric acid, citricacid esters such as triethylcitrate, polyalcohols), co-solvents (e.g.,triacetin, propylene carbonate, ethyl lactate, propylene glycol, oleicacid, dimethylisosorbide, stearyl alcohol, cetyl alcohol, cetostearylalcohol, glyceryl behenate, glyceryl palmitostearate), surfactants,buffering agents, lubricating agents, humectants, preservatives,colorants and flavorants. Capsules may be hard or soft. Examples of hardcapsules include ConiSnap®, DRcaps®, OceanCaps®, Pearlcaps®, Plantcaps®,DUOCAP™, Vcaps® and Vcaps® Plus capsules available from Capsugel®. Hardcapsules may be prepared, for example, by forming two telescopingcapsule halves, filling one of the halves with a fill comprising acompound of formula (I) or pharmaceutically acceptable salt thereof, andsealing the capsule halves together. The fill may be in any suitableform, such as dry powder, granulation, suspension or liquid. Examples ofsoft capsules include soft gelatin (also called softgel or soft elastic)capsules, such as SGcaps®. Soft capsules may be prepared, for example,by rotary die, plate, reciprocating die or Accogel® machine method. Inembodiments, the capsule may be a liquid-filled hard capsule or asoft-gelatin capsule.

Tablets can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared bycompressing in a suitable machine a compound of formula (I) orpharmaceutically acceptable salt thereof in a free-flowing form such asa powder or granules, optionally mixed with a binder, lubricant, inertdiluent, preservative, surface-active or dispersing agent. Moldedtablets can be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletscan be optionally coated or scored and can be formulated so as toprovide sustained, extended, delayed or controlled release. Methods offormulating such sustained, extended, delayed or controlled releasecompositions are known in the art and disclosed in issued U.S. patents,including but not limited to U.S. Pat. Nos. 4,369,174, 4,842,866, andthe references cited therein. Coatings, for example enteric coatings,can be used for delivery of compounds to the intestine (see, e.g., U.S.Pat. Nos. 6,638,534, 5,217,720, 6,569,457, and the references citedtherein). In addition to tablets, other dosage forms, such as capsules,granulations and gel-caps, can be formulated to provide sustained,extended, delayed or controlled release.

In another embodiment, the pharmaceutical composition is formulated forparenteral administration. Examples of a pharmaceutical compositionsuitable for parenteral administration include aqueous sterile injectionsolutions and non-aqueous sterile injection solutions, each containing,for example, anti-oxidants, buffers, bacteriostats and/or solutes thatrender the formulation isotonic with the blood of the intendedrecipient; and aqueous sterile suspensions and non-aqueous sterilesuspensions, each containing, for example, suspending agents and/orthickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example, sealed ampules or vials, and can bestored in a freeze dried (lyophilized) condition requiting only theaddition of a sterile liquid carrier, such as water, immediately priorto use. In one embodiment, the pharmaceutical composition is formulatedfor intravenous administration.

In embodiments, the pharmaceutical composition further includes apharmaceutically acceptable excipient. A pharmaceutically acceptableexcipient may be any substance, not itself a therapeutic agent, used asa carrier, diluent, adjuvant, binder, and/or vehicle for delivery of atherapeutic agent to a patient, or added to a pharmaceutical compositionto improve its handling or storage properties or to permit or facilitateformation of a compound or pharmaceutical composition into a unit dosageform for administration. Pharmaceutically acceptable excipients areknown in the pharmaceutical arts and are disclosed, for example, inRemington: The Science and Practice of Pharmacy, 21^(st) Ed. (LippincottWilliams & Wilkins, Baltimore, Md., 2005). As will be known to those inthe art, pharmaceutically acceptable excipients can provide a variety offunctions and can be described as wetting agents, buffering agents,suspending agents, lubricating agents, emulsifiers, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, andsweeteners. Examples of pharmaceutically acceptable excipients includewithout limitation: (1) sugars, such as lactose, glucose and sucrose;(2) starches, such as corn starch and potato starch; (3) cellulose andits derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, andhydroxypropylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; and (22) other non-toxic compatible substances employedin pharmaceutical formulations.

In embodiments, the pharmaceutical composition can include at least oneadditional active agent. The active agent may be an antineoplastic,chemotherapeutic, cytotoxic, immunomodulator, radiotherapeutic or anyother agent capable of inducing apoptosis, sensitizing a cell toapoptosis, modulating protein kinase or treating neoplasm, tumor orcancer. Examples of the active agent include: (1) antimetabolites, suchas cytarabine, fludarabine, 5-fluoro-2′-deoxyuiridine, gemcitabine,4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-hydroxyrneth 1cyclopent-2-enyl)-1H-pyrimidin-2-one (RX-3117), hydroxyurea ormethotrexate; (2) DNA-fragmenting agents, such as bleomycin, (3)DNA-crosslinking, agents, such as chlorambucil, cisplatin,cyclophosphamide and nitrogen mustard; (4) intercalating agents such asadriamycin (doxorubicin) and mitoxantrone; (5) protein synthesisinhibitors, such as L-asparaginase, cycloheximide, puromycin anddiphtheria toxin; (6) topoisomerase I poisons, such as camptothecin andtopotecan; (7) topoisomerase II poisons, such as etoposide (VP-16) andteniposide; (8) micro-tubule-directed agents, such as colcemid,colchicine, paclitaxel, vinblastine and vincristine; (9) kinaseinhibitors such as flavopiridol, staurosporin and7-hydroxystaurosporine; (10) enzyme poly ADP ribose polymerase (PARP)inhibitors such as olaparib, veliparib, rucaparib, niraparib, andtalazoparib (11) polyphenols such as quercetin, resveratrol,piceatannol, epigallocatechine gallate, theaflavins, flavanols,procyanidins, betulinic acid and derivatives thereof; (12) hormones suchas glucocorticoids and fenretinide; (13) hormone antagonists, such astamoxifen, finasteride and LHRH antagonists; (14) death receptoragonists, such as tumor necrosis factor a (TNF-α), tumor necrosis factorβ (TNF-β), LT-β (lymphotoxin-β), TRAIL (Apo2L, DR4 ligand), CD95 (Was,APO-1) ligand, TRAMP (DR3, Apo-3) ligand, DR6 ligand and fragments; (15)immune checkpoint inhibitors; (16) anti-programmed cell death 1 (PD-1)receptor antibodies or anti-programmed cell death ligand 1 (PD-L1)antibodies; (17) immune checkpoint inhibitors (CTLA-4); and derivativesthereof.

In another embodiment, the amount of the compound of formula (I) orpharmaceutically acceptable salt in the pharmaceutical composition isbetween about 0.1% and about 5% by weight. In another embodiment, theamount is between about 0.5% and about 2.5% by weight.

4.7 Methods of Administration

In any of the methods provided herein, administration of the compound orpharmaceutical composition may be via any accepted mode known in theart, such as orally or parenterally. The term “parenterally” includeswithout limitation subcutaneously, intravenously, intramuscularly,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, by intraosseous injection and by infusion techniques. Inone embodiment, the compound or pharmaceutical composition isadministered orally. In another embodiment, the compound orpharmaceutical composition is administered parenterally. In anotherembodiment, the compound or pharmaceutical composition is administeredintravenously. In another embodiment, the compound or pharmaceuticalcomposition is administered intratumorally.

In one embodiment, the compound or pharmaceutical composition isadministered orally at a dose or dosage as disclosed herein, such as inSection 4.2.

The dose level can be adjusted for intravenous administration. In suchcase, the compound or pharmaceutical composition can be administered inan amount of between about 0.01 μg/kg/min to about 100 μg/kg/min.

4.8 Combination Therapy

In any of the methods of treating or preventing a tumor provided herein,the method may also include the step of administering one or moreadditional anti-tumor agent or radiation to the subject. In oneembodiment, the method includes administering radiation to the subject.In another embodiment, the method further includes administering one ormore additional anti-tumor agent to the subject.

The additional anti-tumor agent or radiation may be administered before,after, or during administration of the compound of formula (I) orpharmaceutically acceptable salt thereof. In one embodiment, theadditional anti-tumor agent or radiation is administered beforeadministration of the compound of formula (I) or pharmaceuticallyacceptable salt thereof. In another embodiment, the additionalanti-tumor agent or radiation is administered after administration ofthe compound of formula (I) or pharmaceutically acceptable salt thereof.In another embodiment, the additional anti-tumor agent or radiation isadministered during administration of the compound of formula (I) orpharmaceutically acceptable salt thereof. In another embodiment, theadditional anti-tumor agent and the compound of formula (I) orpharmaceutically acceptable salt thereof are formulated into apharmaceutical composition for concurrent administration.

The term “anti-tumor agent,” as used herein, refers to any agent usefulfor treating or preventing tumor. Examples of an anti-tumor agentinclude the active agents described in Section 4.6. In one embodiment,the additional anti-tumor agent is selected from antimetabolites,DNA-fragmenting agents, DNA-crosslinking agents, intercalating agents,protein synthesis inhibitors, topoisomerase I inhibitors, topoisomeraseII inhibitors, microtubule-directed agents, kinase inhibitors (e.g.,tyrosine kinase inhibitors), polyphenols, hormones, hormone antagonists,death receptor agonists, enzyme poly ADP ribose polymerase (PARP)inhibitor, immune checkpoint inhibitors, anti-programmed cell death 1(PD-1) receptor antibodies and anti-programmed cell death ligand 1(PD-L1) antibodies. In another embodiment, the additional anti-tumoragent is a PD-1 receptor antibody. In another embodiment, the additionalanti-tumor agent is pembrolizumab. In another embodiment, the additionalanti-tumor agent is nivolumab. In another embodiment, the additionalanti-tumor agent is tremelimumab. In another embodiment, the additionalanti-tumor agent is ipilinumab. In another embodiment, the additionalanti-tumor agent is a combination of nivolumab and ipilinumab. Inanother embodiment, the additional anti-tumor agent is a combination ofpembrolizumab and tremelimumab.

4.9 Process of Making RX-5902

U.S. Pat. No. 8,314,100 discloses a process of converting3-amino-2-chloro-6-fluoroquinoxaline to RX-5902 in 3 steps, throughintermediates 3-amino-6-fluoro-2-methoxyquinoxaline and ethylN-(6-fluoro-2-methoxyquinoxaline-3-yl)carbamate.

During the small batch process of the reaction, a demethylated impurity(based on Mass Spec data) was detected and purification was required forits removal. Furthermore, the small batch process requires intermediatesto be concentrated to dryness. Thus, the process is unsatisfactory forcommercial scale production. Therefore, there is a need to provide animproved process amenable to scale up in fixed equipment to allow forefficient commercial production. For example, the process was improvedby removing the concentration to dryness, substituting some of thehalogenated solvents with non-halogenated solvents and by improving thevolume inefficient recrystallization of Compound 1 in the small batchmanufacturing process.

Below details the small batch production and the improved productionprocess of RX-5902.

4.9.1 Small Batch Production of 1.5 kg of RX-5902 Drug Substance

Scheme 1. Small Batch Synthetic Process for the Production of RX-5902

The present invention provides a method of preparing a compound offormula (I).

or pharmaceutically acceptable salt thereof on a commercial scale, by:(a) reacting 3-amino-6-fluoro-2-methoxyquinoxaline with ethylchloroformate in an organic solvent in the presence of a base to form amixture; (b) distilling the mixture while adding ethyl acetate to form asuspension; (c) filtering the suspension to isolateethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl) carbonate; and (d) reactingthe ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl) carbonate with1-(3,5-dimethoxyphenyl) piperazine hydrochloride in a second organicsolvent in the presence of a second base.

A 1.5 kg scale current good manufacturing practice (cGMP) production of4-(3,5-dimethoxyphenyl)-N-(7-fluoro-3-methoxyquinoxalin-2-yl)piperazine-1-carboxamide(RX-5902) was conducted. The initial production afforded 1.318 kg ofRX-5902. However, when release testing was performed, an unspecifiedimpurity at RRT 0.57 was found to be out of specification (result: 0.82%vs. limit 0.50%). The impurity was later identified by mass spectrometryto correspond to what is believed to be demethylated RX-5902. A basewash rework procedure was successfully developed and implemented,ultimately affording 1.128 kg (designated as batch 35444A).

Embodiments of the method may include: (e) reacting3-amino-2-chloro-6-fluoroquinoxaline with sodium methoxide in an organicsolvent in the presence of a base to form a mixture; (f) adding water tothe mixture of step (e) to form a solution; (g) cooling the solution toa temperature of about 15-20° C. to form a suspension; and (h) filteringthe suspension of step (g) to isolate3-amino-6-fluoro-2-methoxyquinoxaline.

In embodiments, the organic solvent in step (a) may be dichloromethane.In embodiments, the base in step (a) may be pyridine. In embodiments,the distilling step (b) may be conducted under atmospheric pressure. Inembodiments, step (c) may be by vacuum filtration. In embodiments, thesecond organic solvent in step (d) may be tetrahydrofuran. Inembodiments, the second base in step (d) may be1,8-diazabicycloundec-7-ene. In embodiments, the steps may be performedin one or more fixed reactors.

4.9.2 Large Scale Synthetic Production of 11 kg of RX-5902

The present invention provides an improved process of preparing RX-5902,which is commercially viable for large scale production. The processimproves the small batch manufacturing process of RX-5902 to allow scaleup in fixed equipment for commercial scale production to significantlyreduce the cost of manufacture. The inventive process removed theconcentration to dryness step, replaced some of the halogenated solventsand improved the volume inefficient recrystallization of Compound 1 usedin the small batch manufacturing process.

Scheme 2 illustrates an improved process for fixed reactors/large scaleproduction of RX-5902. As shown in Scheme 2, Embodiments of the methodcan include preparing RX-5902 by reactingethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl)carbonate (Compound 3) with1-(3,5-dimethoxyphenyl) piperazine hydrochloride in an organic solventin the presence of a base until the reaction is complete as indicated byHPLC. In an embodiment, the organic solvent may be tetrahydrofuran. Inan embodiment, the base may be 1,8-diazabicycloundec-7-ene (DBU).

Embodiments of the method can include reacting Compound 2 with ethylchloroformate in an organic solvent in the presence of a base to formCompound 3. In an embodiment, Compound 2 may be dissolved in the organicsolvent in the presence of the base before ethyl chloroformate is slowlyadded to the solution. In an embodiment, the organic phase may beextracted with DI water and the organic phase may be distilled underatmospheric pressure to remove the organic solvent. In an embodiment,ethyl acetate may be added during the distillation process. In anembodiment, Compound 3 in solid form may be collected by vacuumfiltration and washed with ethyl acetate and dried. In an embodiment,the organic solvent may be dichloromethane. In an embodiment, the basemay be pyridine.

Embodiments of the method can include converting Compound 1 to3-amino-6-fluoro-2-methoxyquinoxaline (Compound 2) with sodium methoxidein tetrahydrofuran until completion. In an embodiment, water may beadded to the solution mixture. In an embodiment, the solution may becooled to a temperature of about 15-20° C. In an embodiment, thereaction mixture may be concentrated through atmospheric distillation toremove tetrahydrofuran and reduce the volume to less than half. In anembodiment, Compound 2 may be washed with water and collected byfiltering.

Embodiments of the method can include a six-step process of makingRX-5902 with 1,2-diamino-4-fluorbenzene as the starting material. In anembodiment, the process utilizes fixed equipment for a scale up processsuitable for cGMP production of RX-5902.

Embodiments of the method can include converting1,2-diamino-4-fluorbenzene to 6-fluoro-1,4-dihdryoquinoxaline-2,3-dione(Compound A) by reacting 1,2-diamino-4-fluorbenzene with oxalic acid.Compound A may be precipitated and collected by vacuum filtration.

Embodiments of the method can include converting Compound A to2,3-dichloro-6-fluoroquinoxaline (Compound B) by refluxing compound Awith excess thionyl chloride in chloroform until completion. In anembodiment, the reaction may be quenched with sodium hydroxide andstirred until the remaining thionyl chloride are decomposed. In anembodiment, chloroform may be distilled away via atmosphericdistillation. In an embodiment, heptane may be added duringdistillation. In an embodiment, Compound B may be filtered and washedwith heptane and dried in a vacuum. In an embodiment, the filtrate (i.e.mother liquor) may be distilled under vacuum to recover additionalCompound B.

Embodiments of the method can include converting Compound B to3-amino-2-chloro-6-fluoroquinoxaline (Compound 1) with ammoniumhydroxide until completion. In an embodiment, Compound 1 may be purifiedby filtering the reaction mixture warm, i.e. at an elevated temperatureof 45±5° C.

In particular, the present invention of the process improvements removedthe concentrate to dryness operations in Steps 2, 4 and 5 (for theproduction of Compound B, Compound 2 and Compound 3, respectively) andimproved the volume efficiency in the purification of Compound 1. Thepresent invention also replaced the chlorinated solvents in Steps 2, 4,and 5 with non-chlorinated solvents.

As detailed below, the procedures to avoid concentrating to dryness weresuccessful.

For Step 2, most of the chloroform was distilled away and then replacedwith heptane which provided a filterable suspension of Compound B.

For Step 3, the purification of Compound 1 was also improved.Specifically, the purification was accomplished by filtering thereaction mixture warm (45±5° C.), which resulted in an improved yield of72.6% in comparison to a yield of 62% using the small batch procedure.Furthermore, the undesired regioisomer was surprisingly reduced toacceptable levels. (See Table 1.)

TABLE 1 Level of Regioisomer Impurity Levels in Various Batches ofRX-5902 RX-5092 Compound 1 Compound 2 Compound 3 Regioisomer BatchRegioisomer Regioisomer Regioisomer (7-F) 35444A† 0.9% 0.7% NT* 0.6%35686A† 0.7% 0.35% NT* 0.3% 35921A† 1.83% ND** ND** 0.08% †Batches35444A and 35686A were made by the prior small batch process asdescribed below in Example 13, and Batch 35921A was prepared by theimproved fixed reactor/large scale process as described below in Example14. NT*—Not Tested ND**—Not Detected

For Step 4, two improvements were realized in making Compound 2. First,the removal of the concentrate to dryness step was accomplished byquenching the reaction into water and then distilling off thetetrahydrofuran followed by filtering of the product. This new work upalso reduced halogenated solvents. The work up avoided the use ofdichloromethane in product extraction.

For Step 5, the concentration to dryness step was removed by atmosphericdistillation of dichloromethane and replacing it with ethyl acetate insitu. The solvent swap provided another easily filterable slurry toisolate the desired product in high purity and better yield.

4.10 Nanoformulations of RX-5902

The present invention provides new nanoformulations of RX-5902 havingimproved oral bioavailability and methods of making nanoformulations ofRX-5902. For example, the present invention provides a method forreducing the particle size of the compound of formula (I), orpharmaceutically acceptable salt thereof, under conditions sufficient toprovide a suspension. In embodiments, the suspension may be made by amilling process. In embodiments, the milling process can be a low-energymilling process, for example, roller milling. In embodiments, themilling process can be a high-energy milling process, for example,high-energy agitator milling. In embodiments, the milling process may behigh-energy agitator milling or roller milling. In embodiments, themilling process may be high-energy agitator milling. In embodiments, thesuspension may have a D50 particle size of about 200 nm or less. Inembodiments, the method may include lyophilization of the suspension toform a powder. In embodiments, the method may include spray drying thesuspension to form a powder.

4.10.1 Reprocessing of RX-5902 Nanosuspension by Low-Energy Milling andLyophilization

Reprocessing of RX-5902 nanosuspension from previous preparations can beused to produce a nanosuspension of RX-5902 for non-GLP use. Prior toprocessing, the nanosuspension was analyzed to determine if extendedstorage had adversely affected either the chemical or the physicalproperties of the Active Pharmaceutical Ingredient (API), in this case,RX-5902, particles. The suspension was processed by roller milling toreduce observed agglomeration to a more acceptable particle-sizedistribution and then lyophilized to produce a dry powder.

Reprocessing the aged suspension successfully produces a dry powder witha particle-size distribution comparable to that of a clinical bath,which had been milled from unprocessed API and immediately lyophilized.Previously manufactured batches were able to be milled to more uniformparticle-size distributions without agglomeration, which indicated thatthe age of the reprocessed nanosuspension might adversely affect themilling efficiency. For example, the milled material that had been usedto make a clinical batch had been reduced to a monomodal, submicrondistribution with a D90 of about 200 nm; whereas, the D90 of thereprocessed material had a lower limit of ˜800 nm.

Lyophilization conditions appear to play a role in the finalparticle-size distribution of the dry powder as well. Research anddevelopment batches that had been dried using a lyophilizer with a −80°C. condenser resulted in more favorable particle-size distributions thandid either the clinical batch or the reprocessed batch. Both were driedwith a condenser of −53° C. The latter two batches were also made inlarger quantities than was the research material, which may indicatethat freezing time is also relevant to the formation of aggregates inthe dry powder formulation.

4.10.2 RX-5902 High-Energy Nanomilling Process Development and SprayDrying Feasibility

Alternative techniques were tested in both the milling and drying ofRX-5902 nanosuspension to enhance the efficiency and scalability of theproduction of the final dried powder. Previously prepared nanosuspensioncan alternatively be reprocessed by high-energy milling and dried usinglyophilization or spray-drying. High-energy agitator milling can be usedinstead of roller milling to prepare the starting nanosuspension. Spraydrying can be used instead of lyophilization to prepare the dry powder.Agitator milling produces a similar nanoparticle size distribution withonly minor adjustment to the suspension formulation. No apparent APIdegradation was observed. Spray drying produced a narrow particle-sizedistribution in the micron-size range, but does not appear to affect theAPI nanoparticle either physically or chemically.

RX-5902 appears to be amendable to both agitator milling andlyophilization or spray drying with no appreciable degradation or lossattributable to either process. The only major change needed totransition the nanosuspension preparation to agitator milling was thedilution of the starting preparation. This is not expected to have anydeleterious effect on dry powder production because the concentration ofthe API in the diluted agitator-milled material is greater than that ofthe final concentration obtained from the original roller millingprocess. Further dilution should be allowable if necessary to affectefficient extraction of the API from the media.

The original dry powder formulation had been developed using 10% RX-5902that had been modified by the addition of poloxamer to provideprotection against aggregation during the freeze drying. Using spraydrying to product the final powder allows for the omission of thedilution, assay, and poloxamer-addition steps of the process. While theparticle-size distribution of the spray-dried powder was significantlylarger than that of the lyophilized material, the measurement reflectedthe size of the microspheres and not of the nanocrystals, which appearto be unaffected by the process.

4.10.3 Alternative RX-5902 Nanoformulations and Processes

Extended milling times caused foaming during the 1.5 kg manufacture ofRX-5902 nanoformulation. The foaming was mitigated by intermittentrefrigeration of the sub-batches during production. The extended millingtimes and intermittent refrigeration produced the same quality ofRX-5902 nanoformulation as produced in smaller-scale batches.

4.10.4 Milling Operations for RX-5902

As particle size reduction is a key parameter for bioavailability ofRX-5902, various methods of reducing particle size can be utilized andoptimized. These methods include Mirconization, Mechanical Milling,Cryogenic Milling, Wet Milling methods (Agitator and Rolling Mills),Microfluidization, and High Pressure Homogenization. Wet-milling methodscan be followed by either a lyophilization or spray drying method toprovide solid form.

An amorphous formulation prepared by methods including Holt MeltExtrusions, Spray Dried Dispersions with an excipient present and SprayCongealing can also be used. All these methods can form an amorphousform that is anticipated to possess improved bioavailability.

A lipid formulation of RX-5902 can also be utilized. The lipidformulation may benefit RX-5902 providing a pre-solubilized orpre-suspended API in an oil phase. A lipid based formulation would alsobe expected to have improved bioavailability.

4.11 RX-5902 Crystal Structure

4.11.1 XRPD Study of RX-5902 Crystals

An X-Ray Powder Diffraction (XRPD) analysis of RX-5902 crystals providedthe major peaks are shown in Table 2. A complete listing of peaks andother parameters is provided in the working examples.

TABLE 2 Major peaks in XRPD of RX-5902 Pos. d-spacing Rel. Int. [°2Th.][Å] [%] 8.558004 10.33244 92.11 14.346660 6.17385 38.83 15.3286805.78046 33.92 15.574780 5.68967 79.32 15.850830 5.59121 62.55 16.9707605.22467 44.44 18.141790 4.88998 67.28 21.479630 4.13705 69.54 21.8370504.07014 31.41 23.658390 3.76076 66.17 24.417090 3.64560 33.76 24.8524303.58272 100.00 27.474790 3.24643 93.49

As shown above, the crystalline form of RX-5902 (designated Form 1) hascharacteristic peaks (degrees 2Theta) is characterized by peaks at 8.56,15.57, 15.85, 18.14, 21.48, 23.66, 24.85, and 27.47. Form 1 RX-5902 canbe further characterized by peaks (degrees 2Theta) at 8.56, 14.35,15.33, 15.57, 15.85, 16.97, 18.14, 21.48, 21.83, 23.66, 24.41, 24.85,and 27.47. The XRPD of Form 1 RX-5902 is also characterized by a tracehaving d-spacing (A) of 10.33, 5.69, 5.59, 4.89, 4.14, 3.76, 3.58, and3.25. The XRPD of Form 1 RX-5902 is further characterized by a tracehaving d-spacing (A) of 10.33, 6.17, 5.78, 5.69, 5.59, 5.22, 4.89, 4.14,4.07, 3.76, 3.65, 3.58, and 3.25.

4.11.2 Polymorph Study

A batch of RX-5902 was characterized by various solid state techniques.The supplied material was a crystalline solid, denoted as Form 1. Thismaterial is non-hygroscopic, stable to exposure to humidity andrepresents a viable form for further development. XRPD analysis of aformulation containing RX-5902 showed peaks consistent with Form 1,suggesting that no form change had occurred during the formulationprocess.

Polymorph screening experiments performed using crystalline andamorphous RX-5902 identified multiple crystalline forms of the API. Manyof these were poorly crystalline in nature and difficult to reproducefor full evaluation. XRPD diffractograms of several of these forms showsimilarities, suggesting them to be structurally related, whilst thevarying amounts of solvent present raise the possibility that these arechannel solvate type structures.

The difficulties encountered in re-preparing various observed solidsmeant that it was not possible to gain a full understanding of thepolymorphic landscape of RX-5902. The propensity of the API tocrystallize in differing forms means that a rigorous crystallizationprotocol will be required in order to ensure reliable preparation ofForm 1. Studies aimed at identifying suitable solvents for such aprocedure suggested diethyl ether, 2-methyl-1-propanol, ethanol and MIBKas good candidates.

5. EXAMPLES

The following examples are presented for illustrative purposes andshould not serve to limit the scope of the disclosed subject matter.

Example 1: In Vitro Metabolism Studies on RX-5902

Two in vitro studies were performed to examine the involvement ofspecific cytochrome P450 (CYP450) isozymes in the in vitro metabolism ofRX-5902. In the first study, the loss of RX-5902 was measured afterincubation with expressed CYP450 isozymes (1A2, 2A6, 2B6, 2C8, 2C9,2C19, 2D6, 3A4 and 3A5). The results indicate loss of RX-5902 after30-minute incubations with CYPs 3A4 (87% loss) and 3A5 (54% loss), butlittle loss with any other CYP isozyme. In the second study, RX-5902 wasincubated with pooled human liver microsomes in the presence of CYP450isozyme-selective chemical inhibitors. The inhibitors tested wereselective for 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5. Markedinhibition of RX-5902 metabolism was observed in the presence of aCYP3A4/5-selective inhibitor (ketoconazole), but no significantinhibition was observed with inhibitors of the other isozymes. Together,these studies suggest that the CYP450-mediated metabolism of RX-5902 isprimarily due to the CYP3A4/5 isozymes, with little metabolism via otherCYP450 isozymes.

The data indicate that RX-5902 metabolism and exposure may beparticularly sensitive to drug-drug interactions with drugs that alterCYP3A4/5 activity. Thus, co-administration of RX-5902 with CYP3A4/5inhibitors may increase plasma concentrations of RX-5902, whileco-administration with CYP3A4/5 inducers may reduce plasmaconcentrations of RX-5902. Thus, there is the potential that subjectsreceiving CYP3A4/3A5 inhibitors may have exaggerated pharmacological ortoxic responses to RX-5902 or that those receiving CYP3A4 inducers mayhave reduced RX-5902 activity.

Example 2: Pharmacokinetics, Safety and Tolerability of RX-5902 inHumans

In a dose-ranging study, the pharmacokinetics (PK), safety andtolerability of RX-5902 at various oral doses were evaluated. Subjectswith advanced or metastatic solid tumors were administered capsulescontaining RX-5902 at daily doses of 25-775 mg once weekly, 250-300 mgthree times a week, 150-300 mg five times a week, or 300-350 mg seventimes a week of RX-5902 for up to 6 dosing cycles. Each cycle consistingof 1-5 doses of RX-5902 per week for 3 weeks followed by 1 week of rest,or 5-7 doses of RX-5902 per week for 4 weeks without any rest per 4-weekcycle. All but one subject had fasted from food for at least 8 hoursbefore administration. One subject received 300 mg RX-5902 in fed state.Plasma concentrations were measured on Days 1 and 15, for 48 hrs, usinga validated LC-MS/MS assay, and noncompartmental pharmacokineticparameters were calculated using Phoenix WinNonlin, Version 6.4.

Pharmacokinetics (PK)

Preliminary PK data after a single administration is presented in FIG. 1(for Subjects #1-11) and Table 3.

TABLE 3 Human PK Data Dose Frequency C_(max) T_(max) T_(1/2) AUC₀₋₂₄AUC₀₋₄₈ mg Subject # Per week Day Food ng/mL hr Hr hr * ng/mL hr * ng/mL25 1 1 1 Fasted 99.1 6  5.8  830 894 50 2 1 1 Fasted 109 1.5 13.2 10271308 100 3 1 1 Fasted 252 2 27.6 1783 2341 150 4 1 1 Fasted 226 6 11.52689 3280 225 5 1 1 Fasted 364 4 12.0 3425 4312 300 6 1 1 Fasted 318 614.6 4679 6141 300 7 1 1 Fasted 452 1.5 15.6 4170 5552 425 8 1 1 Fasted660 2 — 9321 14673 575 9 1 1 Fasted 707 4 — 6825 10098 775 10 1 1 Fasted487 1.5 — 3541 5012 775 11 1 1 Fasted 654 4  9.7 6925 8126 300 12 1 1Fed 779 4 11.9 8615 10749 250 13 3 1 Fasted 394 2 14.0 3975 5211 250 133 15 Fasted 403 2 — 4812 7774 300 14 3 1 Fasted 288 6 10.3 3848 4555 30014 3 15 Fasted 301 2 — 3049 4143 150 15 5 1 Fasted 227 2 — 2152 — 150 155 15 Fasted 347 1 — 3721 — 200 16 5 1 Fasted 337 4  8.5 2752 — 200 16 515 Fasted 440 2 — 4034 — 300 17 5 1 Fasted 317 2 — 3798 — 300 17 5 15Fasted 460 1.5 — 3840 — 300 18 5 1 Fasted 549 1.5 — 4607 — 300 18 5 15Fasted 536 1 — 4878 — 300 19 5 1 Fasted 419 4 — 3855 — 300 19 5 15Fasted 190 2 — 300 20 5 1 Fasted 624 2 300 21 7 1 Fasted 713 4 — — — 30021 7 15 Fasted 1250 6 — — — 300 22 7 1 Fasted 374 2 — 2986 3178 300 23 71 Faasted 276 4 — — — 300 24 7 1 Fasted 391 1.5 — — — 350 25 7 1 Fasted750 1.5 — 4819 4957

Compared to the subject dosed with 300 mg in the fasted state,significantly higher exposure was observed in the subject dosed with 300mg in a fed state (Table 3).

RX-5902 sometimes displayed an apparent, short lag time (0.25 hour),usually followed by a steep, rising plasma phase. T_(max) was somewhatvariable, being observed from 1 to 6 hours after dosing. After T_(max),a short distribution phase was often observed, followed by the apparentterminal phase. Usually, over 75% of AUC_(0-t) (0-48 hours) was observedby 24 hours. Apparent terminal T_(1/2) ranged from 5.8 to 27.6 hours,but most individual values were near the mean value of 13.0 hours.C_(max) and AUC_(0-t) (0-48 hours) increased fairly linearly with dose.AUC_(last) increased in a dose-proportional manner overall, but C_(max)increased in a less than proportional manner. Clinical C_(max) and AUCranges for various doses and dose frequencies are shown in Table 3.

Safety and Tolerability

The most frequently reported adverse events were mild nausea, vomitingand fatigue. The results show that at the tested dose levels, RX-5902 iswell tolerated.

Example 3: Protocol for Evaluating Efficacy of RX-5902 in XenograftModels of Cancer

The efficacy of RX-5902 in human cancer xenograft mouse models isexamined. Female nude mice (nu/nu, Harlan or CRL: NU(NCr)-Foxn1^(nu),Charles River), 9-10 weeks old, with a body weight (BW) range of 15-30 gon day 1 of the study, are fed ad libitum water (reverse osmosis, 1 ppmCl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0%crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice are housedon irradiated Enrich-o'cobs™ Laboratory Animal Bedding in staticmicroisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and40-60% humidity. The study complies with the recommendations of theGuide for Care and Use of Laboratory Animals with respect to restraint,husbandry, surgical procedures, feed and fluid regulation, andveterinary care.

Various human tumor cell lines (e.g., HCT116, HT29, H460, H69, Caki-1,CaSki, MiaPaca2, BxPC3 and Colo 205 cells [ATCC, Manassas, Va., USA])are cultured according to ATCC's instruction. The tumor cells arecultured in tissue culture flasks in a humidified incubator at 37° C.,in an atmosphere of 5% CO₂ and 95% air.

The cells are harvested during exponential growth and re-suspended withphosphate buffered saline. Each test animal receives a subcutaneous(s.c.) injection of 5×10⁶ tumor cells into the right flank and tumorgrowth is monitored as the average tumor size approaches the targetrange of 80-300 mm³. When tumors reach the target size mice arerandomized into several groups (n=10-20) and treatment with variousregimens of RX-5902 or a positive (e.g., gemcitabine) or negativecontrol (e.g., vehicle, saline) is initiated. Tumors and body weightsare measured regularly until the study is terminated.

Tumors are measured in two dimensions using calipers, and volume iscalculated using the formula:

${{Tumor}\mspace{14mu}{Volume}\mspace{14mu}\left( {mm}^{3} \right)} = \frac{w^{2} \times l}{2}$where w=width and l=length, in mm, of the tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Treatment begins on Day 1 in eight groups of mice (n=10-20/group) withestablished subcutaneous tumors of a particular cell line. Each group istreated according to the study design. All doses are adjusted per bodyweight. Animals in each group are divided for efficacy and samplingpurposes. There are also groups designated to vehicle control and notreatment for sampling purposes.

On Day 1 of the study, all animals from no-treatment group are sampledfor tumor and whole blood. Additionally, four animals from other groupsare sampled 2, 8 and 24 hours post first dose and 24 hours post seconddose. Mice are sacrificed by terminal cardiac puncture under isofluoraneanesthesia. Full blood volume is collected into a tube containinglithium heparin anticoagulant. Each blood sample is processedindividually for plasma using lithium heparin as anticoagulant for PBMC.The tumors are collected and divided in halves where one part is fixedfor 24 hours in 10% neutral buffered formalin (NBF), and thentransferred to 70% ethanol and the other half is snap frozen. The plasmaand tumor frozen samples are stored at −80° C.

Treatment efficacy is determined using data from Day 15. The MTV (n),the median tumor volume for the number of animals, n, on Day 15, isdetermined for each group. Percent tumor growth inhibition (% TGI) isdefined as the difference between the MTV of the designated controlgroup (vehicle administration) and the MTV of the drug-treated group,expressed as a percentage of the MTV of the control group:% TGI=[1−(MTV_(drug treated)/MTV_(control))]×100The data set for TGI analysis includes all animals in a group, exceptthose that die due to treatment-related (TR) or non-treatment-related(NTR) causes. An agent that produces at least 60% TGI in this assay isconsidered to be potentially therapeutically active.

Animals are monitored individually for tumor growth until Day 71. Thestudy protocol specifies a tumor growth delay assay based on the mediantime to endpoint (TTE) in a treated group versus the control group. Eachanimal is euthanized for tumor progression (TP) when its tumor reachesthe 2000 mm³ volume endpoint. The time to endpoint (TTE) for each mouseis calculated with the following equation:

${T\; T\; E} = \frac{{\log_{10}\left( {{endpoint}\mspace{14mu}{volume}} \right)} - b}{m}$where b is the intercept and m is the slope of the line obtained bylinear regression of a log-transformed tumor growth data set. The dataset is comprised of the first observation that exceeds the studyendpoint volume and the three consecutive observations that immediatelyprecede the attainment of the endpoint volume. Any animal that does notreach endpoint is euthanized at the end of the study and assigned a TTEvalue equal to the last day of the study (71 days). In instances inwhich the log-transformed calculated TTE precedes the day prior toreaching endpoint or exceeds the day of reaching tumor volume endpoint,a linear interpolation is performed to approximate TTE. Any animaldetermined to have died from treatment-related (TR) causes is assigned aTTE value equal to the day of death. Any animal that dies fromnon-treatment-related (NTR) causes is excluded from TTE analysis.

On Day 71, MTV (n) is defined as the median tumor volume of the numberof animals, n, that survives to the last day and whose tumors has notreached the volume endpoint. Any animal determined to have died fromtreatment-related (TR) causes is to be assigned a TTE value equal to theday of death. Any animal that dies from nontreatment-related (NTR)causes is to be excluded from the analysis. Treatment outcome isevaluated from tumor growth delay (TGD), which is defined as theincrease in the median TTE for a treatment group compared to the controlgroup:TGD=T−Cexpressed in days, or as a percentage of the median TTE of the controlgroup:

${\%\mspace{14mu} T\; G\; D} = {\frac{T - C}{C} \times 100}$where T=median TTE for a treatment group, and C=median TTE for thecontrol group.

Treatment efficacy is also determined from the number of regressionresponses. Treatment may cause partial regression (PR) or completeregression (CR) of the tumor in an animal. In a PR response, the tumorvolume is 50% or less of its D1 volume for three consecutivemeasurements during the course of the study, and equal to or greaterthan 13.5 mm³ for one or more of these three measurements. In a CRresponse, the tumor volume is less than 13.5 mm³ for three consecutivemeasurements during the course of the study. Any animal with a CRresponse on the last day of the study is additionally classified as atumor-free-survivor.

For toxicity assessments, animals are weighed daily for the first fivedays of the study and twice weekly thereafter. The mice are observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity are recorded when observed.

Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen resulting in greatertoxicity is considered above the maximum tolerated dose (MTD). A deathis classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or if due to unknown causesduring the dosing period or within fourteen days of the last dose. Adeath is classified as non-treatment-related (NTR) if there is noevidence that death was related to treatment side effects.

Prism 6.05 (GraphPad) for Windows is employed for statistical andgraphical analyses. MTV values for multiple groups are compared with thenon-parametric Kruskal-Wallis test and a post hoc Dunn's multiplecomparison test. The two-tailed statistical analyses are conducted atP=0.05. Prism reports results as non-significant (ns) at P>0.05,significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”)at 0.001<P≤0.01 and extremely significant (“***”) at P≤0.001. Becausestatistical tests are tests of significance and do not provide anestimate of the size of the difference between groups, all levels ofsignificance are described as either significant or non-significantwithin the text of this report.

Survival is analyzed by the Kaplan-Meier method, based on TTE values.The log rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests determine thesignificance of the difference between the overall survival experiences(survival curves) of two groups, based on TTE values. The Kaplan-Meierplot and statistical tests share the same data sets, and exclude anyanimals that are recorded as NTR deaths. A scatter plot is constructedto show TTE values for individual mice, by group; this plot shows NTRdeaths, which are excluded from all other figures. Group mean tumorvolumes are plotted as functions of time. When an animal exits the studybecause of tumor size or TR death, its final recorded tumor volume isincluded with the data used to calculate the median volume at subsequenttime points. Tumor growth curves are truncated after two TR deaths occurin the same group. Group mean Body Weight (BW) changes over the courseof the study are graphed as percent change, ±SEM, from D1. Tumor growthand BW change curves are truncated after more than half the assessablemice in a group exits the study.

Example 4: Efficacy of RX-5902 in Renal Cell Carcinoma Xenograft Model

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human renal tumor xenografts (Caki-1) wasexamined. Tumor growth delay was measured as the increase in median timeto the endpoint tumor volume in a treated group compared to a vehicletreated group. Efficacy of RX-5902 was determined using two differentdosing schemas: weekly dosing at 20-160 mg/kg for 4 weeks (FIG. 3), or50-70 mg/kg daily (5 days on/2 days off) for 3 weeks (FIG. 2). Weeklydosing of RX-5902 at 160 mg/kg resulted in a 75% TGD (P<0.001) (FIG. 3).Daily administration of RX-5902 resulted in dose-dependent TGI (80 and96%; Day 21) and TGD (68 and 104%, P<0.001) (FIG. 2), and extended theoverall survival of the animals at both doses (P<0.0001) (data notshown). At the dose of 70 mg/kg daily, 6/10 animals demonstrated partialtumor regressions and 1/10 a complete tumor regressions. RX-5902 did notresult in a reduction in body weight gain, treatment related deaths, orclinical observations in either of the dosing schemas. Sunitinib(positive control in this study; 60 mg/kg; daily for 21 days) resultedin TGD for both in vivo studies validating the Caki-1 model herein.These data support the potential therapeutic activity of RX-5902 inrenal cell cancers and extending survival. The results also suggest thatmore frequent dosing, with lower daily doses, in humans may be a moreeffective administration schedule for RX-5902 in renal cancer.

Results of Xenograft studies (described in Examples 4-9) are summarizedin Table 4.

TABLE 4 Anti-tumor Activity of Orally Administered RX-5902 in MiceXenograft (TGI %* or TGD{circumflex over ( )}) Dose MDA- SK- (mg/kg)Schedule Caki-1 MiaPaca-2 Colo205 MB-231 A2780 OV3 A549 160 QWK75%{circumflex over ( )}  0%* 44%*; 43%* 30%* 39%* 320 QWK 13%* 65%*;43%* 26%* 82%* 600 QWK 33%* 83%*; 75%* 26%* 165%* 40 5ON/2OFF 6%{circumflex over ( )} 50 5ON/2OFF 68%{circumflex over ( )} 83%{circumflex over ( )} 60 5ON/2OFF 70 5ON/2OFF 104%{circumflex over( )}  339%{circumflex over ( )} 49%{circumflex over ( )} {circumflexover ( )}denotes TGD %; *denotes TGI %. In some instances both valuesare reported.

Example 5: Efficacy of RX-5902 in Pancreatic Cancer Xenograft Model

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human pancreas tumor xenografts (MiaPaca-2)was examined. Tumor growth was measured in a treated group compared to avehicle treated group. The results show marked efficacy with 50 or 70mg/kg RX-5902 administered 5 days a week (See Table 4), suggesting thatdaily dosing in humans may be an effective treatment schedule forRX-5902 in pancreatic cancer.

Example 6: Efficacy of RX-5902 in Colorectal Cancer Xenograft Model

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human colorectal tumor xenografts (Colo205)was examined. Tumor growth was measured in a treated group compared to avehicle treated group. The results show marked efficacy with 320 and 600mg/kg weekly RX-5902 administration (See Table 4), suggesting thatRX-5902 may be an effective treatment in colorectal cancer.

Example 7: Efficacy of RX-5902 in Breast Cancer Xenograft Model and Roleof Phosphorylated P68

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human breast tumor xenografts (MDA-MB-231) wasexamined. Tumor growth was measured in a treated group compared to avehicle treated group. The results show marked efficacy with 160, 320,and 600 mg/kg weekly RX-5902 administration (See Table 4; FIG. 12), andextending the survival in treated mice (FIG. 13). These results suggestthat RX-5902 may be effective in treating breast cancer and extendingsurvival.

It was also determined whether phosphorylated p68 on Tyr593 played a keyrole in RX-5902's ability to inhibit cancer cell growth by knocking downp68. p68-siRNA efficiently down-regulated the expression ofphosphorylated p68 on Tyr593 as well as p68 in the triple-negative (TN)breast cancer cell line, MDA-MB-231. Exposure of p68-siRNA-transfectedcells to the IC₅₀ concentration of RX-5902 protected MDA-MB-231 cellsfrom the cytotoxic effects of RX-5902, indicating that phosphorylatedp68 on Tyr593 is a key molecule for RX-5902's cytotoxic effects.

Example 8: Efficacy of RX-5902 in Cisplatin-Resistant Ovarian CancerXenograft Model

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human ovarian tumor xenografts (A-2780) wasexamined. Tumor growth was measured in a treated group compared to avehicle treated group. The results show marked efficacy with 160, 320,and 600 mg/kg weekly RX-5902 administration (See Table 4). Similarresults were obtained in another model of ovarian tumor xenograft(SK-OV3) at 40 and 70 mg/kg RX-5902 given daily (Table 4). These resultssuggest that RX-5902 may be an effective treatment in human ovariancancer.

Example 9: Efficacy of RX-5902 in Non-Small Cell Lung Cancer XenograftModel

Following the protocol described in Example 3, the effect of RX-5902 ontumor growth in mice with human non-small cell lung tumor xenografts(A549) was examined. Tumor growth was measured in a treated groupcompared to a vehicle treated group. The results show efficacy with 160,320, and 600 mg/kg weekly RX-5902 administration (See Table 4),suggesting that RX-5902 may be an effective treatment in lung cancer.

Example 10: Efficacy of RX-5902 in Syngeneic MC38 Murine Colon CancerXenograft Model

Following the methods described below, the effect of RX-5902 on tumorgrowth in a syngeneic model using female C57BL/6 mice with MC38 murinecolon cancer was examined. Tumor growth was measured in a treatmentgroup compared to a control (vehicle treated) group (see Table 5 belowfor dosing schema and treatment regimen). These data demonstrate thatthe addition of RX-5902 to a programmed death receptor 1 (PD-1)inhibitor, RMP1-14, had an additive effect in the inhibition of tumorgrowth (90% RX-5902 alone, 93% RMP1-14 alone, versus 99% in combinationof two agents [P<0.01 versus control group]. Combination of the twoagents also resulted in higher number of mice (6 mice) with partialregression and complete regression with 4 animals showing tumor freesurvival, compared to 4 animals with partial regression and completeregression in the RMP1-14 alone group with 2 showing tumor freesurvival. All results were obtained without any adverse effects to themice in the combination group. This study demonstrates that thecombination of RX-5902 and a PD-1 inhibitor result in a significantreduction in tumor growth, resulting in partial and complete responsesand tumor free survival in mice, in the absence of any adverse event.

TABLE 5 Drugs and Treatment Schedule: Regimen 1 Regimen 2 Gr. N Agentmg/kg Route Schedule Agent mg/kg Route Schedule 1# 10 Vehicle — po (5/2)× 3 — — — — 2 10 RX-5902  84.34 po (5/2) × 3 — — — — 3 10 anti-PD-1RMP1-14 100* ip biwk × 2 — — — — 4 10 RX-5902  84.34 po (5/2) × 3anti-PD-1 RMP1-14 100* ip biwk × 2 #Control Group *μg/animal

The present study consisted of five groups (n=10 per group) of femaleC57BL/6 mice bearing subcutaneous MC38 tumors (mean tumor volume range:66-68 mm3) on Day (D1) of the study, when dosing began. Vehicle wasadministered orally (p.o.). RX-5902 was administered p.o. at 84.34 mg/kg(70 mg/kg active dose). Anti-PD-1 (RMP1-14) was administered at 100μg/animal, intraperitoneally (i.p.). Group 1 mice served as controls andreceived PBS (vehicle) five days on, two days off for three cycles((5/2)×3). Group 2 received RX-5902 (5/2)×3. Group 3 received anti-PD-1twice weekly for two weeks (biwk×2). Group 4 received RX-5902 (5/2)×3and anti-PD-1 biwk×2. The study endpoint was a tumor volume of 1500 mm³or 45 days, whichever came first. Tumor measurements were taken twiceweekly until Day 45 with individual animals exiting the study uponreaching the tumor volume endpoint.

Partial treatment outcome was based on percent tumor growth inhibition(% TGI), defined as the percent difference between Day 28, chosen forthe TGI analysis, median tumor volumes (MTVs) of treated and controlmice. The results were analyzed and were deemed statisticallysignificant at P≤0.05. A treatment that produced at least 60% TGI wasconsidered to have potential therapeutic activity. Additionally,efficacy was determined from tumor growth delay (TGD), a measure of theincrease in the median time to endpoint (TTE) in a treatment, comparedto the control group. Response was additionally evaluated based on thenumber of study survivors, partial regression (PR) and completeregression (CR) responses, and logrank significance of differences insurvival. Tolerability of the various treatments was assessed by bodyweight (BW) measurements and frequent observation for clinical symptomsand treatment-related (TR) side effects.

On Day 28, the median tumor volume for the control Group 1 was 1226 mm³,with an individual tumor range of 14 to 1800 mm³. Seven tumors in thecontrol Group 1 reached the volume endpoint with a median TTE of 29.7days, establishing a maximum T-C of 15.3 days (52% TGD) in the 45-daystudy. The control TTE ranged from 26.9 to 45.0 days. The variability ofthe control group decreased the likelihood of achieving statisticalsignificance. The MTV of the three survivors was 14 mm³ and there weretwo PRs.

Administration of RX-5902 resulted in a significant 90% TGI (P<0.05,Mann-Whitney). This therapy produced a median TTE of 44.8 days or anon-significant 51% TGD. Five animals remained on D45 with an MTV of 14mm³ and there were three PRs. Treatment with anti-PD-1 led to asignificant 93% TGI (P<0.05, Mann-Whitney). This therapy resulted in sixsurvivors and an assigned TTE of 45.0 days and the maximum possible 52%TGD. Results were not significant. The MTV on D45 was 14 mm³ and therewas one PR and three CRs; two animals with the latter ended the study astumor-free survivors (TFS).

Combination therapy with RX-5902 and anti-PD-1 resulted in a 99% TGI.This outcome was significant compared to the control group (P<0.01,Mann-Whitney), but did not significantly differed from anti-PD-1monotherapy. This dual therapy was assigned a median TTE of 45.0 days orthe maximum possible 52% TGD. All ten animals survived with an MTV of 14mm³. There was one PR and five CRs, four of which were TFS. Results weresignificant when compared to controls (P<0.01, log rank) as well asmonotherapy (P<0.05 for both comparisons, log rank).

Example 11: Effect of RX-5902 on β-Catenin Dependent ATPASE Activity ofY593 Phosphorylated P68 and Expression of Genes Regulated by β-Catenin

Materials and Methods

Cell Culture and Antibodies

MDA-MB-231, SK-MEL-28, and WI-38 cells were obtained from ATCC(Manassas, Va., USA) and were cultured according to the vendor'sinstructions. Anti-p68 antibody and antiY593-p68 antibody were purchasedfrom Cell Signaling (Danvers, Mass.) and Abcam (Cambridge, Mass.),respectively. Antibodies against β-actin, cyclin D1, p-c-Jun and c-Myc,were purchased from Santa Cruz (Dallas, Tex.). Anti-phospho-tyrosineantibody and HRP conjugated GAPDH antibody were obtained from CellSignaling (Danvers, Mass.). Recombinant β-catenin and p68 protein werepurchased from Creative Biomart (Shirley, N.Y.) and Origene (Rockville,Md.), respectively.

Recombinant Proteins

Recombinant β-catenin was used without further treatment whereasrecombinant p68 protein was either used as p68 or phosphorylated byc-Abl for filter binding assay. Recombinant c-Abl was obtained fromAbcam (Cambridge, Mass.).

Drug Treatment

RX-5902 was dissolved in DMSO to prepare a stock solution of 2 mM. Thestock solution was stored at −20° C. and diluted with medium to prepareworking concentrations.

Identification of RX-5902 Binding Proteins

MDA-MB-231 cells were plated onto 6 well plates and treated with RX-5902at various concentrations (0, 0.1, 1 and 10 μM) for one hour. Cells werelysed with p-MER buffer containing protease/phosphatase inhibitors onice. Cell lysates were treated with thermolysin protease (1:1,500 ratio)for 10 min at RT and the reaction was stopped by addition of 0.5 M EDTAsolution. The reaction mixtures were separated on a 10% SDS-PAGEvisualized by Coomassie staining. After identifying several candidateproteins from mass spectrometry sequencing analysis, the protein whichinteracted with RX-5902 was confirmed by western blot analysis.

Filter Binding Analyses

Filter binding studies have been previously described elsewhere (Coombset al., Proc. Natl. Acad. Sci. USA, 75:5291-5295 (1978). Briefly,recombinant p68 RNA helicase with/without tyrosine phosphorylation wasadded to the ³H-labeled RX-5902 (10 Ci/mmol) with PBS. ³H-labeledRX-5902 was synthesized from Quotient Bioresearch (Cardiff, UK). Afterincubation at room temperature for 30 minutes, the binding mixtures wereloaded onto a nitrocellulose membrane. The membrane was washed fivetimes with PBS, and then dried by vacuum. The amounts of RX-5902 boundto p68 with/without phosphorylation of p68 were determined by ³Hscintillation counting. The same procedure was done with ³H-labeledRX-5902 alone without addition of p68 RNA helicase and sample p68 RNAhelicase alone without addition of the ³H-labeled RX-5902 as background³H scintillation counting. The binding percentages of p68 to thecompound were calculated and plotted against concentrations. Thedissociation constant (Kd) was estimated by the concentration at 50% ofp68 bound to RX-5902 and calculated by linear regression analysis.

ATPase Assay

ATPase activity was determined by measuring the released inorganicphosphate during ATP hydrolysis using a direct colorimetric assay (Shinet al., Cancer Res., 67:7572-7578 (2007); Yang et. al., Cell,127:139-155 (2006)). A typical ATPase assay was carried out in 50 μlreaction volumes, containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mMMgCl₂, 5 mM DTT, ˜1-2 μg of appropriate substrate, 4 mM ATP, and 10 μlof helicase. The ATPase reactions were incubated at 37° C. for 30minutes. After incubation, 1 ml of malachitegreen-molybdenum reagent wasadded to the reaction mixture, and reactions were further incubated atroom temperature for exactly 5 minutes. The absorption (A) at 630 nm wasthen measured. The concentrations of inorganic phosphate were determinedby matching the A_(630 nm) in a standard curve of A_(630 nm) vs. knownphosphate concentrations. The proteins, p68 or phospho-p68, used forthis assay were prepared in-house similar to the procedure reportedpreviously (Yang et al., Protein Expr. Purif., 35:327-333 (2004)). Thepercentage of inhibition by defining the ATPase activity of phospho-p68without RX-5902 as zero percent inhibition was calculated and IC₅₀ ofRX-5902 was calculated by non-linear regression analysis using KalediaGraph software program (Synergy Software, Reading, Pa.).

Western Blotting

Protein mix or cell lysates were separated by SDS-PAGE and transferredto PVDF membrane. The membrane was blocked by blocking buffer (1×TBSTcontaining 5% BSA) at room temperature for 1 hour. After a brief wash,the membrane was incubated with primary antibody in blocking buffer at4° C. overnight. After incubation in primary antibody, the membrane waswashed with 1×TBST three times and subsequently incubated with HRPconjugated secondary antibody in blocking buffer at room temperature for1 hour. The membrane was again washed three times and visualized by ECLsystem (Thermo Scientific, Rockford, Ill.).

Results

Well-established target identification method DARTS assay (Lomenick etal., Proc. Natl. Acad. Sci. USA, 106:21984-21989 (2009)) was used tofind target proteins that would interact with RX-5902 in cancer cells.DARTS method indicated that a band with mobility around 60 kDa wasprotected from the protease cleavage upon interaction with RX-5902 (FIG.4). To identify this protected protein, the bands along with controlwere sliced out, and analyzed with LC-MS/MS by ProtTech (Phoenixville,Pa.). Western blot analysis was carried out using antibodies againstseveral potential candidates from LC-MS/MS analysis that have a similarmobility in SDS-PAGE to confirm the protected protein by RX-5902treatment. Clearly, this protected band was recognized by the antibodyagainst p68 RNA helicase (FIG. 5), indicating that RX-5902 may interactwith p68 RNA helicase in cells and protect p68 from degradation bythermolysin. To verify the interaction of RX-5902 with p68, ³H-labeledRX-5902 was used. The interaction of ³H-labeled RX-5902 with recombinantp68 protein and the in vitro tyrosyl phosphorylated recombinant p68protein was probed by filter binding assays (Coombs et al., supra).Through western blot analysis, it was confirmed that p68 wasphosphorylated on a tyrosine residue (FIG. 6). A filter binding assayclearly showed RX-5902 interacted with the Y593 phospho-p68 with anestimated Kd around 19 nM, but RX-5902 did not interact withunphosphorylated p68 in the filter binding studies (FIG. 7; squares andtriangles represent duplicate experiments).

The effect of RX-5902 on ATPase activity of p68 was investigated. To dothis, ATPase activity of recombinant p68 in the presence of RX-5902 andtotal RNA extracted from yeast were measured. RX-5902 did not affectRNA-dependent ATPase activity of p68 RNA helicase at 0.2 μM and even athigh concentrations such as 20 μM, RNA-dependent ATPase activity wasinhibited by less than 30% (FIG. 8), indicating RX-5902 had very littleeffect on RNA-dependent ATPase activity of p68. This data could confirmthat RX-5902 did not interact with unphosphorylated p68 in the filterbinding assay (FIG. 7).

The results show that the β-catenin dependent ATPase activity ofphospho-p68 was largely diminished in the presence of RX-5902 at 0.2 μM(FIG. 9). The experiment was repeated at lower concentrations of RX-5902to calculate IC₅₀. The IC₅₀ of RX-5902 for the inhibition of β-catenindependent ATPase activity was calculated to be 61 nM (FIG. 10),indicating that RX-5902 potentially disrupts the phospho-p68/β-catenininteraction.

Since RX-5902 directly binds to Y593 phospho-p68, it was hypothesizedthat treatment of cells with RX-5902 would interfere with thephospho-p68/β-catenin interaction and consequently affect the expressionof several growth associated genes, including p-c-Jun, c-Myc and cyclinD, which are regulated byphospho-p68/β-catenin interaction. Thus, cyclinD1 and c-Myc expression, as well as phosphorylation of c-Jun in cellstreated with RX-5902, were analyzed. Cancer cell lines, SK-MEL-28 andMDA-MB-231, and normal fetal lung fibroblasts, WI-38, were used.Although 20 nM RX-5902 did not change protein levels, 70 nM RX-5902 ledto a decrease in expression of both cyclin D1 and c-Myc and a decreasein c-Jun phosphorylation in SK-MEL-28 without changing the level oftotal p68 protein. Similar changes in protein levels were detected inMDA-MB-231 with both 20 nM and 70 nM RX-5902. RX-5902 did not result inany significant change in cyclin D1 or c-Myc expression or c-Junphosphorylation in WI-38 cells (FIG. 11), even at 70 nM. The resultsdemonstrate that treatment of cancer cells with RX-5902 resulted in thedownregulation of the expression of certain genes which are known beregulated by the β-catenin pathway, such as c-Myc, cyclin D1 andp-c-Jun. Therefore, the study indicates that inhibition of Y593phospho-p68 helicase-β-catenin interaction by direct binding of RX-5902to Y593 phospho-p68 RNA helicase may contribute to the anti-canceractivity of this compound.

Example 12: Efficacy, Safety and Tolerability of RX-5902 in Humans

The efficacy, safety and tolerability of RX-5902 at various doses andfrequencies are evaluated. In the Phase 1 portion of the study, subjectswith advanced solid tumor malignancies are enrolled. During the Phase 1portion of the study, the maximum tolerated dose (MTD) or recommendedphase 2 dose (RP2D) and schedule are determined and the pharmacokineticsof RX-5902 characterized in eligible subjects. Subjects with advancedmalignant tumors are administered capsules containing RX-5902 at dosesof 125-1050 mg/day 1, 3, 5, or 7 day(s) a week for 3 weeks with 1 weekoff during each 4 week cycle, or 4 weeks without a week off during the 4week cycle. Dose escalation begins with an accelerated design treating 1subject per dose (Simon et al., J. Natl. Cancer Inst., 89(15):1138-47(1997) followed by a standard 3+3 design using a modified Fibonaccisequence after the occurrence of a single related Grade 2 or greateradverse event.

In the Phase 2 portion, subjects are enrolled in 1 of 2 diagnosisgroups: triple negative breast cancer or platinumresistant/refractory/relapsed ovarian cancer. The Phase 2 portion of thestudy uses the dose and schedule identified in the Phase 1 and follows a2-stage design. An interim analysis is conducted when 10 responseevaluable subjects in each tumor indication are enrolled and have hadthe opportunity to complete a minimum of 4 cycles of therapy or havediscontinued therapy due to progressive disease. In the second stage ofPhase 2, enrollment for a disease group proceeds if at least 2 responsesare observed within the first 10 response-evaluable patients enrolled inthat disease group. Approximately 40 additional subjects are enrolled ineach of the disease indications. In the second stage of Phase 2, overallresponse rate and progression free survival rate is further evaluated inthe disease groups that continue beyond the first stage. Table 6summarizes the potential dose and schedule, but this schedule may changebased on safety and tolerability data.

TABLE 6 Dosing Schedule Total Dose Doses per Daily dose weekly doseWeeks of Group Week (mg) (mg) Dosing* 1 5 150 750 3 2 3 250 750 3 3 3300 900 3 4 5 150 750 3 or 4 5 5 200 1000 3 or 4 6 5 250 1250 3 or 4 7 5300 1500 3 or 4 8 5 350 1750 3 or 4 9 5 400 2000 3 or 4 10 7 200 1400 3or 4 11 7 300 2100 3 or 4 12 7 350 2450 3 or 4 13 7 400 2800 3 or 4*indicates weeks of dosing out of a 4-week cycle

Example 13: Small Scale Production of RX-5902 (Preparation of Batch35444a) Example 13A Production of Compound A

A 100-L reactor was charged with 1,2-diamino-4-fluorobenzene (1.75 kg,13.8 mol, ChemiK), oxalic acid (1.25 kg, 13.9 mol), 11.4 kg water and3.8 kg conc. hydrochloric acid (3 M HCl solution, 3.28 equiv HCl). Thedark mixture was heated at reflux (95-100° C.) for ˜25 h. An aliquot (1mL) was taken while stirring and neutralized with saturated NaHCO₃solution (30 mL) to a pH of 8. HPLC analysis showed that the startingmaterial was completely consumed.

The mixture was removed from heating, allowed to cool to roomtemperature, and then cold water (14 kg) was added. A dark solidprecipitated and was collected by vacuum filtration. The filter cake waswashed with cold water (12 kg), followed by isopropyl alcohol (IPA, 9.4kg). The wet caked (3.4 kg) was then dried overnight in a vacuum oven(50° C.) to give 2.38 kg (96% molar yield) of6-fluoro-1,4-dihydroquinoxaline-2,3-dione (Compound A) as a blue-graysolid with an HPLC purity of 97.0%.

Example 13B Production of Compound B

To a 100-L reactor was added Compound A (2.38 kg, 13.2 mol),dimethylformamide (DMF, 0.25 kg) and CHCl₃ (37.0 kg). Then, thionylchloride (SOCl₂, 4.9 kg, 41.2 mol) was added to maintain temperature<25° C. The mixture was then refluxed at 55-60° C. After 23 h, a samplewas taken for HPLC analysis. HPLC analysis showed complete consumptionof starting material but what appeared to be ˜2% of an intermediate, inaddition to 98% 2,3-dichloro-6-fluoroquinoxaline (Compound B). Anadditional amount of SOCl₂ (494 g) and DMF (25 g) was added and themixture refluxed an additional 1 h. The HPLC was unchanged so thereaction mixture was cooled to room temperature and deionized (DI) water(8.8 kg) was added slowly into the reaction mixture, which resulted inevolution of heat and gas. This was followed by slow addition of 0.5 MNaOH (8 kg). The entire quench was stirred 13 h to help decompose theexcess SOCl₂. The organic layer was washed with 4×8 kg water followed by8×16 kg water washes.

The organic phase (in a 50 L reactor) was treated with 40 g of activatedcarbon and agitated for 35 minutes. The activated carbon was removed byfiltration and the batch was concentrated to dryness. The resultingsolid was dried in a vacuum oven (46° C.) to give 2.62 kg (94% molaryield) of Compound B with an HPLC purity of 98.0%.

Example 13C Production of Compound 1

To a 100-L reactor was added 2,3-dichloro-6-fluoroquinoxaline (CompoundB, 1.30 kg, 6.00 mol), acetonitrile (32.0 kg) and ammonium hydroxide(11.9 kg). The mixture was stirred at 50° C. for 28 h. Approximately 4%of unreacted Compound B was observed. An additional 1.2 kg of ammoniumhydroxide was added and the mixture was stirred an additional 20 h at50° C. HPLC analysis showed no remaining starting material. The batchwas cooled to 20° C. and the solids were vacuum filtered using Büchnerfilter, rinsed with ACN/water and dried in a vacuum oven overnight at50° C. to yield 972 g of crude 3-amino-2-chloro-6-fluoroquinoxaline(Compound 1). The reaction was repeated at the same scale a second time,affording 954 g of crude Compound 1. The combined crops (1.92 kg) wereslurried in 27 kg water for 30 min, then filtered and dried at 50° C. ina vacuum for ˜40 h, affording 1.90 kg of desalted, crude Compound 1. Aportion of the material (0.63 kg) was recrystallized by dissolution inacetonitrile (55 kg) at 75-80° C. and cooling to ambient temperature.The cake was isolated by filtration and dried in a vacuum oven at 5° C.for ˜20 h. A 0.286 kg portion of purified Compound 1 was obtained (HPLC:98.9%, 1.0% regioisomer). A large percentage of solids were left in thereactor for the next iteration of the recrystallization process.Starting again from 0.63 kg of crude material and this time utilizing 76kg acetonitrile (to account for solids from the first portion), arecovery of 0.686 kg was obtained (HPLC: 98.8%, 0.9% regioisomer).Finally, the last portion (0.63 kg) was recrystallized as before from 55kg acetonitrile to afford 0.505 kg of isolated product (HPLC: 98.8%,0.9% regioisomer). The total yield was 1.48 kg (62% overall yield) ofCompound 1.

Example 13D Production of Compound 2

To a 100-L round bottom flask was added3-amino-2-chloro-6-fluoroquinoxaline (Compound 1, 1.48 kg, 7.49 mol) andTHF (26.7 kg). Then 25 wt % NaOCH₃ in MeOH (12.1 kg, 56.0 mol, 7.5equiv) was added so as to maintain temperature of 20±10° C. The mixturewas stirred at room temperature for 2 h. HPLC showed the startingmaterial was consumed. The solution was concentrated under reducedpressure to approximately 21 L and then partitioned overnight betweendichloromethane (DCM, 34 kg) and water (34 kg). The aqueous layer wasseparated and then the organic layer was further diluted with anotherportion of DCM (34 kg). The organic layer was washed with water untilthe pH of the aqueous layer reached 5 (4×19 kg washes). The organiclayer was concentrated to give a solid. The solids were slurried in DCM(2.1 kg), filtered and the wet cake was washed with DCM (1×5 kg). Thewet cake was dried under vacuum at 44° C. overnight to afford 1.06 kg(73.3% yield) of 3-amino-6-fluoro-2-methoxyquinoxaline (Compound 2),HPLC: 97.0% (0.7% regioisomer).

Example 13E Production of Compound 3

To a 100-L reactor was added 3-amino-6-fluoro-2-methoxyquinoxaline(Compound 2, 1.06 kg, 5.48 mol), DCM (21.4 kg) and pyridine (0.63 kg,7.97 mol). The mixture was stirred at room temperature and then ethylchloroformate (872 g, 8.0 mol, 1.46 equiv) was slowly added whilemaintaining the temperature below 30° C. After 18.5 h at roomtemperature, the reaction was found to be incomplete. More ethylchloroformate (174 g, 1.6 mol) and pyridine (126 g, 1.6 mol) were added.The reaction was then complete by HPLC analysis after an additional 28.5h mixing at room temperature. The organic phase was extracted withdeionized water until a pH of 5.8 was obtained (4×16.1 kg). The organiclayer was dried with magnesium sulfate (970 g), filtered andconcentrated to dryness. Ethyl acetate (3.2 kg) was then charged and theresulting solids were collected via vacuum filtration. The wet cake waswashed with ethyl acetate (1.3 kg) and dried overnight under vacuum at43° C. to yield ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl) carbonate(Compound 3) (HPLC purity 100%, 1.116 kg, 76.7% yield).

Example 13F Production of RX-5902

To a 100-L reactor was added 1-(3,5-dimethoxyphenyl) piperazine HCl(DMPP, 1.588, 6.14 mol), ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl)carbonate (Compound 3, 1.12 kg, 4.22 mol) and THF (30.6 kg). The mixturewas stirred at ambient temperature for 15 mins and1,8-diazabicycloundec-7-ene (DBU, 2.444 kg, 14.7 mol) was added. Themixture was mixed at reflux (˜66° C.) for ˜4 h, then sampled forreaction completion (HPLC result: 1.1% Compound 3 remaining). Thereaction was deemed complete. The mixture was concentrated under vacuumto ˜11 L volume. The solution was diluted with DCM (20.2 kg) and washedwith ˜12 kg of 1M HCl. The organic layer was further washed with 4×10 kgportions of water until the pH of the aqueous waste layer was 5-6. Theorganic layer was dried over anhydrous magnesium sulfate (1.2 kg) andthe filtered through a Büchner funnel. The cake was rinsed with 5 kg ofDCM and the filtrate was concentrated under vacuum to a final volume of6 L. Heptane (0.868 kg) was then added and the slurry mixed at 10-15° C.for ˜16 h. The slurry was filtered and washed with heptane (2.62 kg).The wet cake (2.45 kg) was then dried in a vacuum oven. After ˜48 h at66° C., IP (In Process) testing showed THF (4506 ppm) and DCM (3250 ppm)to be above the limits. After an additional 96 h at 66° C., only the THF(1141 ppm) was still above the limit. After an additional 24 h at 66°C., the residual THF dropped to 770 ppm. Finally, after another 24 h at67° C., the THF level dropped to a passing level (679 ppm). The materialwas then removed from the oven to afford RX-5902 as an off-white solid(1.514 kg, 81.2% yield). HPLC: RRT 0.57 impurity of 0.82% above the speclimit of NMT 0.50%.

RRT 0.57 as identified by mass spectrometry is believed to correspond toa demethylated form of RX-5902.

Example 13G Purification of RX-5902

To a 100-L reactor was added RX-5902 from Example 13F (1.318 kg, 2.99mol) and DCM (17.5 kg). The solution was washed with ˜7 kg of 0.5 M NaOH(aq). The organic layer was further washed with 4×5.3 kg portions ofwater until the pH of the aqueous waste layer was 5-6. The organic layerwas dried over anhydrous magnesium sulfate (0.6 kg) and then filteredthrough a Büchner funnel. The cake was rinsed with 2 kg of DCM and thefiltration was concentrated under vacuum to a final volume of 4 L.Heptane (2.70 kg) was added and the slurry mixed at 10-15° C. from ˜15h. The slurry was filtered and washed with heptane (1.86 kg). The wetcake was then dried in a vacuum oven. After ˜48 h at 55° C., IP (InProcess) testing showed passing levels of all solvents. The material wasremoved from the oven to afford RX-5902 as an off-white solid (1.14 kg,86.5% recovery).

Example 14: Fixed Reactors/Large Scale Production of RX-5902(Preparation of Batch 35921A)

Below details the production of 10.96 kg of cGMP batch for RX-5902 wasconducted under the following improved process conditions using fixed35, 100, 124, 200 and 300 gallon glass, stainless and Hastelloy linedreactors and stainless steel or Hastelloy Aurara filters.

Example 14A Production of Compound A

A 35 gallon reactor was charged with 1,2-diamino-4-fluorbenzene (7.50kg, 59.46 mol, ChemiK), oxalic acid (5.35 kg, 59.42 mol) and 3 M HClsolution (95.8 kg water and 37.8 kg conc. hydrochloric acid). The darkmixture was heated at reflux (100±5° C.) for ˜21 h. An aliquot (1 mL)was taken while stirring and neutralized with saturated NaHCO₃ solution(30 mL) to a pH of 8. HPLC analysis showed that the starting materialwas completely consumed.

The mixture was removed from heating, allowed to cool to ambienttemperature and then cold water (55 kg) was added. A dark solidprecipitated and was collected by vacuum filtration. The filter cake waswashed with cold water (50.0 kg) followed by Isopropyl Alcohol (IPA,39.14 kg). The wet cake (11.43 kg) was then dried overnight in a vacuumoven (50±5° C.) to give 10.3 kg (96% molar yield) of6-fluoro-1,4-dihdryoquinoxaline-2,3-dione (Compound A) as a blue-graysolid with an HPLC purity of >99%). The process was repeated on theidentical scale to provide 9.95 kg (>99%).

Example 14B Production of Compound B

To a 200 gallon reactor was added6-fluoro-1,4-dihdryoquinoxaline-2,3-dione (Compound A, 20.25 kg, 112.41mol), DMF (2.20 kg) and chloroform (310.3 kg). Thionyl chloride (40.95kg) was added while maintaining the temperature <25° C. The mixture wasrefluxed at 50-55° C. After 21 h, a sample was taken for HPLC analysis.HPLC analysis showed complete consumption of starting material with97.7% (by area) of Compound B. The reaction mixture was cooled toambient temperature (25±5° C.) and DI water (64.7 kg) was added slowlyinto the reaction mixture, which resulted in evolution of heat and gas.Next, 0.5 M NaOH (64.7 kg) was added slowly. The entire quench wasstirred for 13 h to help decompose the excess SOCl₂. The organic layerwas washed with 8×32.8 kg water.

The organic phase (in a 200 gallon reactor) was concentrated byatmospheric distillation until approximately 3 gallon remained. Asdistillation was progressing, heptane (69.5 kg) was added to thedistillation pot. Distillation continued until pot temperature exceeded70° C. (70.3° C.). The pot was cooled to 10-15° C. and stirred for 12 h.The slurry was filtered and washed with heptane (2×16.0 kg). Theresulting wet solid (20.65 kg) was dried in a vacuum oven without heatfor 25 h at 45±5° C. to give 14.80 kg (64.5% yield) of2,3-dichloro-6-fluoroquinoxaline (Compound B) with an HPLC purity of97.7%.

Due to the low yield, the mother liquor was reprocessed. The motherliquor was distilled under vacuum until approximately 30 gallons hadbeen removed (˜12 gallons remained in the pot). The jacket was set to40° C. Once distillation was complete, the suspension was cooled to10-15° C. (actual 13.2° C.) and stirred for over 3 h. The slurry wasfiltered and washed with heptane (2×16.0 kg). The resulting wet solid(10.50 kg) was dried in a vacuum oven at 45±5° C. for over 18 h to give3.62 kg of 2,3-dichloro-6-fluoroquinoxaline (Compound B, second lot)with an HPLC purity of 98.4%. The total yield was 75.5% (18.45 kg).

Example 14C Production of Compound 1

To a 200 gallon reactor was added 2,3-dichloro-6-fluoroquinoxaline(Compound B, 18.45 kg, 85.01 mol), acetonitrile (448.60 kg) and ammoniumhydroxide (169.80 kg). The mixture was stirred at 50° C. for nearly 24h. HPLC analysis showed no remaining starting material. The batch wascooled to 45±5° C. and stirred for nearly 25 h. The solids were vacuumfiltered, washed with water (2×14.0 kg) and acetonitrile (2×36.1 kg).The wet cake (16.70 kg) was dried in a vacuum overnight at 50° C. for 49h to yield 3-amino-2-chloro-6-fluoroquinoxaline (Compound 1, 12.20 kg,72.7% yield). Compound 1 was obtained (HPLC: 97.86%, 1.83% regioisomer).

Example 14D Production of Compound 2

To a 200 gallon glass-lined reactor was added3-amino-2-chloro-6-fluoroquinoxaline (Compound 1, 12.10 kg, 61.23 mol)and THF (212.20 kg) followed by 25 wt % NaOCH₃ in MeOH (91.80 kg) so asto maintain the temperature at 20±10° C. The mixture was stirred atambient temperature for ˜4 h. HPLC showed the starting material wasconsumed. Water (43.50 gal) was added while keeping the internaltemperature <35° C. The solution was concentrated through atmosphericdistillation until ˜55 gallons remained (˜63 gallons were removed). Thesolution temperature was cooled to 15-20° C. and stirred for ˜18 h. Theslurry was filtered and washed with water (2×8.0 gallon). The resultingwet solid was dried in a vacuum oven at 50±5° C. for almost 96 h toafford 7.40 kg (62.6% yield) of 3-amino-6-fluoro-2-methoxyquinoxaline(Compound 2) with an HPLC purity of 99.1%; regioisomer was not detected.

Example 14E Production of Compound 3

To a 100 gallon reactor was added 3-amino-6-fluoro-2-methoxyquinoxaline(Compound 2, 7.40 kg, 38.30 mol), DCM (144.6 kg) and pyridine (3.6 kg).The mixture was stirred at ambient temperature and then ethylchloroformate (6.9 kg, 63.58 mol) was slowly added while maintaining thetemperature <30° C. After 21.5 h at ambient temperature, the reactionwas found to be complete. The organic phase was washed with DI wateruntil a pH of 5.8 was obtained (3×16.4 gallons). The organic layer wasdistilled under atmospheric pressure until ˜16 gallons remained. Ethylacetate (47.4 kg) was added during the distillation. The suspension wascooled to 10-15° C. and stirred for 22 h. The solids were collected byvacuum filtration and washed with ethyl acetate (2×8.3 kg) and dried(7.8 kg wet) overnight under vacuum at 40±5° C. to affordethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl) carbonate Compound 3 (7.30kg, 71.8% yield). HPLC: 100%; regioisomer was not detected).

Due to the low recovery, the mother liquor from above was concentratedunder vacuum to approximately half of its original volume (17 L). Thesolids were filtered and rinsed with ethyl acetate (2×1.5 L). The wetcake (1.6 kg) was dried at 40±5° C. for 36 h to afford additional 1.35kg of Compound 3 (E-166). The total yield was 85.1% (8.65 kg).

Example 14F Production of RX-5902

To a 200 gallon reactor was added 1-(3,5-dimethoxyphenyl) piperazine HCl(MVP) (12.50 kg, 48.31 mol),ethyl-N-(6-fluoro-2-methoxyquinoxaline-3-yl) carbonate (Compound 3, 8.65kg, 32.61 mol) and THF (249.6 kg). The mixture was stirred at ambienttemperature for 15 minutes and DBU (17.60 kg, 115.6 mol) was added. Themixture was mixed at reflux (˜66° C.) for ˜4 h and cooled to 20±10° C. Asample analyzed by HPLC showed the reaction was complete (HPLC result:0.6% Compound 3 remaining versus IP test limit of NMR 2.0%). The mixturewas concentrated under vacuum to ˜86 L volume. The solution was dilutedwith DCM (159.70 kg) and washed with 1M HCl (8.9 kg conc. HCl in 24.1gallon water). The organic layer was further washed with water (4×30.0gallons) until the pH of the aqueous waste layer was 5-6. The organiclayer was further washed with) 0.5 N sodium hydroxide (2.9 kg of 50%sodium hydroxide and 18.6 gallon of water) and washed with water (3×25.0gallons) until the pH of the aqueous waster layer was 5-6. The organiclayer was dried over magnesium sulfate (9.5 kg) and filtered through aBüchner funnel. The cake was rinsed with DCM (19.5 kg) and the filtratewas then concentrated under vacuum with DCM (19.5 kg). The filtrate wasconcentrated under vacuum to a final volume of 47 L. Heptane (7 kg) wasadded and the slurry was mixed at 10-15° C. for ˜16 h. The slurry wasfiltered and washed with heptane (21 kg). The wet cake (15 kg) was driedin a vacuum over at 60° C. for ˜48 h. IP testing showed THF (10,800 ppm)and DCM (15,395 ppm) to be above the spec limits. After an additional 86h at 60° C., all solvent levels passed the residual solventspecifications. The material was removed from the oven to afford RX-5902as an off-white solid (10.965 kg, 76.2% yield).

Example 15: RX-5902 Nanoformulation Process for 1.3 KgBatch—Reprocessing of Previously Prepared RX-5902 Example 15A 100 mg/gNanosuspension of RX-5902 Example 15A1—Sub-Batch 1

YTZ® Grinding Media (9 kg) was washed with cleanser solution (Alconox)and rinsed with purified water (Ricca Chemical Company). The cleansersolution was prepared by mixing 5 mL of cleanser and 500 mL of purifiedwater. The media was evenly divided between three heat-resistantcontainers. Each container was enclosed in an autoclave bag with thepermeable side of the bag covering the opening of the container. ATuttnauer 2540EA Electronic Table-Top Autoclave was sterilized using avalidated sterilization cycle (250° F. for 45 minutes with 35 minutesdrying time, see PSSOP 50042 “Operation, Maintenance and Clearing of theTuttnauer 2540EA Electronic Table-Top Autoclave) and the containers ofmedia were placed in the oven to dry at 110° C. overnight.

The following supplies and labware was cleaned with the cleansersolution and transferred into the cleanroom: Milling vessels (×3), Stirplates, Funnels, Disposable spatulas, Weigh containers, Magneticstirrers, Magnetic stirrer retriever and Transfer pipettes. Ahydrothermograph was set up in the manufacturing suite and the humidityand temperature were recorded. A balance was set up in the isolator andthe daily verification was conducted.

Poloxamer 407, NF (Spectrum) was weighed out and charged to each of thecontainers. To vessel A was charged 10.68 g, to vessel B was charged10.64 g and to vessel C was charged 10.70 g. To each of the threevessels was charged Water for Injection, USP (WFI). To vessel A wascharged 299.57 g, to vessel B was charged 300.62 g and to vessel C wascharged 301.19 g. A stir bar was placed into each of the three vesselsand a stir plate was used to mix until the Poloxamer 407, NF was visiblydissolved into the WFI.

To each of the three containers was added by funnel RX-5902(Pfanstiehl). To vessel A was charged 133.16 g, to vessel B was charged133.19 g and to vessel C was charged 133.41 g. The stir plate wasemployed to mix the contents of the containers until RX-5902 was visiblydispersed. The stir bar was removed from each of the three containersemploying a magnetic retriever. The contents of one container wascharged by funnel into a milling vessel. The threads of the millingvessel were inspected to ensure the threads are free from the millingmedia. The lid of the milling vessel was tightened and sealed to preventleakage.

The exterior of the milling vessel was decontaminated and removed fromthe isolator. A piece of reflective material was attached to the millingvessel, and the milling vessel was attached to the roller mill. The millwas activated and the rotational speed was adjusted until the cascadingmedia inside of the vessel achieved an angle-of-break of between 45 to60 degrees from the horizontal (visually determined). A tachometer wasemployed to measure the rotational speed of the milling vessel. Each ofthe three containers was transferred to a milling vessel and preparedusing the same general procedure as described above.

Each of the three milling vessels was roller milled for 18.75 hours at90 RPM (rotations per minute). After the roller milling was complete,the exterior of the vessels were decontaminated and transferred into thelaminar-flow hood. The hydrothermograph was transferred into the samemanufacturing suite to accompany the milling vessels. A sterile pipettewas employed to take a 1 mL sample from each milling vessel and thesamples were transferred to vials.

The three samples were analyzed for particle-size distribution by laserdiffraction. The acceptance criteria for the sample was D90<1 μm(replicates and average) and a monomodal distribution profile (i.e. thedistribution contains one main peak with only a slight secondary peakallowed). The results for each of the vessels were as follows:

D90 D90 D90 D90 Vessel (replicate 1) (replicate 2) (replicate 3)(Average) A 0.21355 μm 0.21216 μm 0.20778 μm 0.21116 μm B 0.16276 μm0.16996 μm 0.17200 μm 0.16824 μm C 0.16087 μm 0.16628 μm 0.16291 μm0.16335 μm

Each of the three vessels met the accepted criteria. The three vesselsfrom sub-batch 1 were closed and stored at 2 to 8° C. until theextraction could be performed.

Example 15A2—Sub-Batch 2

A second sub-batch was made in an identical manner. Vessels were chargedas follows:

Poloxamer 407, Vessel NF (Spectrum) WFI RX-5902 D 10.64 g 299.95 g132.94 g E 10.62 g 300.61 g 133.32 g F 10.65 g 299.73 g 133.32 g

Each of the three milling vessels was roller milled for 19.25 hours at90 RPM (rotations per minute). After the roller milling was complete,the exterior of the vessels were decontaminated and transferred into thelaminar-flow hood. The hydrothermograph was transferred into the samemanufacturing suite to accompany the milling vessels. A sterile pipettewas employed to take a 1 mL sample from each milling vessel and thesamples were transferred to a vials.

The three samples were analyzed for particle-size distribution by laserdiffraction. The acceptance criteria for the sample was D90<1 μm(replicates and average) and a monomodal distribution profile (i.e. thedistribution contains one main peak with only a slight secondary peakallowed). The results for each of the vessels were as follows:

D90 D90 D90 D90 Vessel (replicate 1) (replicate 2) (replicate 3)(Average) D 0.26110 μm 0.25660 μm 0.25724 μm 0.25831 μm E 0.30640 μm0.29270 μm 0.32984 μm 0.30965 μm F 0.43902 μm 0.42497 μm 0.43438 μm0.43279 μm

Each of the three vessels met the accepted criteria. The three vesselsfrom sub-batch 2 were closed and stored at 2 to 8° C. until theextraction could be performed.

Example 15A3—Sub-Batch 3

A third sub-batch was made in an identical manner. Vessels were chargedas follows:

Poloxamer 407, Vessel NF (Spectrum) WFI RX-5902 G  10.68 g, 300.63 g133.89 g H 10.65 g 300.48 g 133.04 g I 10.71 g  300.90 g. 116.19 g

Each of the three milling vessels was roller milled for 19.5 hours at 90RPM (rotations per minute). The roller milling was stopped, and theexteriors of the vessels were decontaminated and transferred into thelaminar-flow hood. The hydrothermograph was transferred into the samemanufacturing suite to accompany the milling vessels. A sterile pipettewas employed to take a 1 mL sample from each milling vessel and thesamples were transferred to a vials.

The three samples were analyzed for particle-size distribution by laserdiffraction. The acceptance criteria for the sample was D90<1 μm(replicates and average) and a monomodal distribution profile (i.e. thedistribution contains one main peak with only a slight secondary peakallowed). The results for each of the vessels were as follows:

D90 D90 D90 D90 Vessel (replicate 1) (replicate 2) (replicate 3)(Average) G 66.69387 μm 0.18573 μm 55.32641 μm 40.73534 μm H 0.29842 μm236.23581 μm 64.05984 μm 100.19800 μm I 75.35052 μm 61.63898 μm 51.83522μm 62.94151 μm

None of the three vessels met the accepted criteria.

Vessels G, H and I were again sealed and returned to the roller mill.The milling was continued for 22.5 hours. The hydrothermograph wastransferred to the same manufacturing suite to accompany the vessel. Asterile pipette was employed to take a 1 mL sample from each millingvessel and the samples were transferred to a vials.

The three samples were analyzed for particle-size distribution by laserdiffraction. The acceptance criteria for the sample was D90<1 μm(replicates and average) and a monomodal distribution profile (i.e. thedistribution contains one main peak with only a slight secondary peakallowed). The results for each of the vessels after additional millingtime were as follows:

D90 D90 D90 D90 Vessel (replicate 1) (replicate 2) (replicate 3)(Average) G 0.17129 μm 0.18762 μm 0.17109 μm 0.13000 μm H 0.17073 μm0.17154 μm 0.17198 μm 0.17142 μm I 0.16724 μm 0.16825 μm 0.17332 μm0.16966 μm

Each of the three vessels met the accepted criteria. The three vesselsfrom sub-batch 3 were closed and stored at 2 to 8° C. until theextraction could be performed.

Example 15B Extraction of the Sub-Batches

The following supplies and labware for the extraction were prepared:Extraction Vessels (×9), Funnels, Tubing, Hose Clamps, In-line airfilter, Transfer pipettes and Filter funnel. The Balance was wiped with70% isopropanol. These supplies were transferred to the cleanroom.

A nitrogen tank was set up in the cleanroom suite by connecting the airfilter to a hose, and the hose was connected to the nitrogen tank. Ahydrothermograph was set up in the manufacturing suite and thetemperature and humidity were recorded.

Nine sub-batches (A-I) were prepared using the extraction process asfollow. An empty collection vessel was weighed and placed under a filterfunnel. The contents of milling vessel A were poured into the filterfunnel and the suspension was extracted using compressed nitrogen. WFIwas charged to the milling vessel. The contents were poured into thefilter funnel and extracted using compressed nitrogen. WFI was chargedto the milling vessel. The contents were poured into the filter funneland extracted using compressed nitrogen. The collection vessel wasweighed to afford the net suspension weight.

The collection vessel was swirled manually to mix the contents. Using asterile transfer pipette, a sample for analytical testing was withdrawnas well as a QA sample. The final weight of the collection vessel wasrecorded.

All sub-batches were stored at 2 to 8° C. until the in-process assay wascompleted.

The yield of the nine sub-batches and the amount available for releasewas as follows:

Sub-batch Process Yield Amount for Release A 93% 1234.94 g B 94% 1247.05g C 95% 1265.06 g D 94% 1251.18 g E 93% 1240.57 g F 93% 1239.88 g G 95%1256.83 g H 96% 1279.70 g I 93% 1241.99 g

The combined total process weight from the nine sub-batches was 11287.95g (94% yield) with 11257.21 g the total amount for release.

Example 15C Lyophilization to Afford 83% RX-5902 Nanoformulation Powder

The amount of RX-5902 in each of the nine sub-batches was calculatedbased on the assay analysis of each sub-batch. The assay amount, finalsuspension weight and amount of RX-5902 in each of the sub-batches wasfound to be as follows:

Sub-batch % Assay Suspension Weight Amount of RX-5902 A 105.6% 1234.94 g130.41 g B 104.1% 1247.05 g 129.82 g C 101.7% 1265.06 g 128.66 g D103.8% 1251.18 g 129.87 g E 103.7% 1240.57 g 128.65 g F 108.0% 1239.88 g133.91 g G 108.3% 1256.83 g 136.11 g H 104.2% 1279.70 g 133.34 g I 94.5%1241.99 g 117.37 g

The total amount of RX-5902 in the nine sub-batches was 1168.14 g.

The following supplies, raw materials, equipment and labware wereprepared for the lyophilization process and transferred to thecleanroom: Balance, Timer, Hydrothermograph, Bulk suspension container,Magnetic stirrer, Stir plate, Magnetic stirrer retriever, Weighingcontainers, Spatulas and Bulk lyophilization trays. The hydrothermographwas set up in the manufacturing suite and recorded the temperature andhumidity conditions.

In the laminar-flow hood, all sub-batches were combined into the bulkcontainer. A stir bar was added and the contents were mixed. To the bulksuspension container was added Poloxamer 407, NF (146.27 g, Spectrum).The contents of the bulk suspension container was mixed until thePoloxamer 407, NF was completely dissolved by visual inspection. Themixing was stopped and the stir bar was removed.

To each of the eight bulk lyophilization trays was added ˜⅛^(th) of thecontents of the bulk suspension container. The lids on each of the eighttrays were closed. The eight trays were transferred to the lyophilizerand the lyophilizer door was closed and sealed. The hydrothermograph wasstopped.

The shelf temperature of the lyophilizer was adjusted to −40° C. and the“Freeze Shelf” function was turned on. The trays were allowed tocompletely freeze over 66.75 hours. The condenser was turned on andallowed to reach −53° C. The shelf temperature was then set to −25° C.and the vacuum setting was set to 250 mTorr for primary drying. After˜18 days, the difference between the last Pirani gauge (284 mTorr) andcapacitance manometer (250 mTorr) readings was <1% of the previousPirani gauge (285 mTorr) and capacitance manometer (250 mTorr) readings.The primary drying was deemed complete.

Over ˜30 minute intervals, the shelf temperature setting was increasedby +5° C. until the shelf setting was 20° C. After ˜4 days, thedifference between the last Pirani gauge (486 mTorr) and capacitancemanometer (500 mTorr) readings was <1% of the previous Pirani gauge (486mTorr) capacitance manometer (500 mTorr) readings. The secondary dryingwas deemed complete. The shelf control and condenser were turned off.The vacuum was turned off and released.

A mortar and pestle were prepared by washing with the cleanser solutionand rinsing with purified water (Ricca Chemical Company). The cleansersolution was prepared by mixing 5 mL of cleanser and 500 mL of purifiedwater. The mortar and pestle were placed in an autoclave bag with thepermeable side of the bag facing upwards. A Tuttnauer 2540EA ElectronicTable-Top Autoclave was sterilized using a validated sterilization cycle(250° F. for 45 minutes with 35 minutes drying time, see PSSOP 50042“Operation, Maintenance and Clearing of the Tuttnauer 2540EA ElectronicTable-Top Autoclave) and the bags containing the mortar and pestle wereplaced in the oven to dry at 250° C. for ˜1 hr. The hydrothermograph wasset up in the manufacturing suite and recorded the temperature andhumidity conditions.

The dried trays were removed from the lyophilizer and transferred intothe manufacturing suite. The holding container was weighed (3911.45 g),sanitized and transferred into a glove-box isolator. The driedlyophilized trays were wiped down and transferred into the glove-boxisolator. The mortar and pestle was employed to break apart thelyophilate into freely flowing powder. The powder was transferred intothe holding container.

The holding containers were weighed (5508.47 g) and sampled. From thetop of the holding container was removed the top sample for homogeneitytesting (1.15 g). From the middle of the holding container was removedthe sample for testing (4.48 g), a QA retain (8.33 g) and a sample formicro testing (11.00 g). From the bottom of the holding container wasremoved a sample for the bottom homogeneity testing (1.21 g). Theholding container was weighed again (5280.12 g) and the hydrothermographwas turned off.

The process batch size was calculated as 1397.02 g (97% Process yield)with a batch size for release as 1368.67 g. The analytical testing forthe batch showed it met all certificate of analysis specifications.

Example 16: High-Energy Milling and Drying Example 16A Productions ofHigh-Energy Milled Material

The Netzsch DeltaVita agitator mill was assembled using the 150-mLrecirculation chamber and a 150-micron outlet screen. About 125 mL (0.5kg) of 0.5-mm YTZ ceramic milling media was added to the chamber and thechamber was cooled to 10° C. using a recirculating chiller. The chamberwas primed for milling by pumping the starting suspension into the millat about 100 mL per minute (48 rpm using MasterFlex size 15 tubing), andby periodically “jogging” the mill by running the agitator for a fewseconds at a time to better disperse the incoming suspension. Onceprimed, the mill was operated at an agitator speed of 500 rpm, or 1.8m/s tip speed. This turned out to be insufficient as the back-pressureat the suspension inlet increased to the point at which the millautomatically shut off. This is typically caused by clogging by APIparticles that either are too large to pass through the screen, or thattend to aggregate in the screen slots. To prevent the pressure build-up,the agitator speed was incrementally increased until the system couldrun without pressure increase, which was at 2,000 rpm (7 m/s).

After 18 minutes, the D90 of the suspension was reduced to about 1micron, which is the informal limit that had been previously used as amaximum particle size for the in-process suspension. After 90 minutes ofmilling, the suspension solidified, an occurrence that is not uncommonwhen reducing particles into the size range that is typical of colloids.About 150 mL of additional purified water was added to the millingreservoir, which brought the API concentration to 20%, and whichliquefied the suspension enough to continue milling. The suspension wasmilled for a total of 240 minutes, at which point the particle-sizedistribution showed a uniform, monomodal, submicron population ofparticles, as shown in FIG. 14. The D10 of the nanosuspension is 0.07284μm, median size is 0.10526 μm, and D90 is 0.15167 μm. The resultingnanosuspension was fluid and uniform, and showed no signs ofdiscoloration or physical change from that which had been observed inthe roller-milled suspensions.

To determine if the crystal structure of RX-5902 had been altered duringmilling, the nanoparticles were tested by DSC and x-ray powderdiffraction (XRPD). The nanoparticles were removed from suspension bycentrifuge filtration (Vivaspin). The particles were washed three timeswith purified water in an effort to remove associated poloxamer. Afterwashing, the particles were dried over silica. DSC analysis, pictured inFIG. 15, showed a slight reduction in melting onset (161° C.) as well asa low temperature (50° C.) thermal event, both of which indicate thepresence of poloxamer (melting point=56° C.) in the isolatednanoparticles. However, no other thermal events indicative of adifferent crystal form of the API were observed. XRPFD analysisconfirmed that the crystal structures of the milled and unmilled APIwere comparable.

Example 16B Productions of High-Energy Milled Lyophilized Material

High-energy milled lyophilized material was prepared lyophilizing thehigh-energy milled material of Example 16A using the lyophilizationmethod of Example 15C.

Example 16C Production of High-Energy Milled Spray-Dried Material

RX-5902 nanosuspension was spray dried with a Buchi B-290 spray dryerusing the parameters outlined in Table 7. The suspension was used as ithad been extracted from the mill, without the addition of any excipientsor purified water.

TABLE 7 Spray-Drying Parameters Parameter Value Nozzle diameter 1.40 mmInlet temperature 100 C. Aspirator 80% Pump rate 20% Q-flow 50

Minimal material loss was observed in the drying chamber of the spraydryer. The collected product was a free-flowing powder that dispersedinto purified water. Particle-size measurements by laser diffractiongave a concise, repeatable distribution as shown in FIG. 16 and themeasurements are shown in Table 8.

TABLE 8 Sizes of RX-5902 nanoparticles Batch Mean Size D10 Median SizeD90 1 3.60752 μm 1.99298 3.42597 5.48444 2 3.41835 1.93907 3.263575.08637 3 3.51103 1.95287 3.34039 5.30013

Microscopy showed the presence of crystalline nanoparticles contained inamorphous spherical microparticles as shown in FIG. 17 and FIG. 18. Nofree crystals or evidence of crystal regrowth was observed, suggestingthat no dissolution or precipitation of API was affected by the elevatedtemperatures of drying.

Example 17: Determination of the Oral Bioavailability of RX-5902Following Intravenous and Oral Administration

In this study, the oral bioavailability of RX-5902 was evaluated in maleSprague-Dawley rats following administration of various formulations.RX-5902 was dosed by intravenous (IV) and oral (PO) routes ofadministration. Four preparations of powders were received fromParticles Sciences (Bethlehem, Pa.) and used to make the dosingsolutions: (Preparation A): Unmilled API (Prepared according to Example13); (Preparation B) 83% GMP lyophilized (Prepared according to Example15); (Preparation C) 83% high-energy milled lyophilized (Preparedaccording to Example 16B); and (Preparation D) 93% spray-dried (Preparedaccording to Example 16C)

Dose levels for each animal were individually determined based on bodyweight and amount of test article administered. For each dose, theappropriate amount of formulation powder was weighed, and then 1 mL ofthe appropriate solvent was added and the total volume was immediatelyadministered, except for the unmilled API where an additional 1 mL ofsolvent was added and dosed to recover all API remaining in the vial.Following dosing, blood samples were collected up to 24 hours post-dose,and plasma concentrations of the test article was determined byLC-MS/MS. Pharmacokinetic parameters were determined using PhonenixWinNonlin (v6.4).

Following IV dosing at 40.7 mg/kg average dose, RX-5902 (83% GMPLyophilized Cake; Group 2) had an average half-life of 10.5±2.46 hours.Its average clearance rate was 0.400±0.0263 L/hr/kg. The average volumeof distribution was 5.60±1.30 L/Kg.

Following PO dosing of unmilled RX-5902 (Unmilled API in 0.36% Poloxamer407 in ultrapure water; Group 1) at 68 mg/kg average dose, maximumplasma concentrations (average of 743±199 ng/mL) were observed between 2and 4 hours post dosing. The average half-life could not be determined;however, the half-life was 3.29 hours for Rat #576. The average exposurebased on the dose normalized AUC_(Last) was 151±25.2 hr*kg*ng/mL/mg. Theaverage oral bioavailability for unmilled RX-5902 (also referred toherein as “Unmilled API”) was 7.62±1.27% at an average dose of 68 mg/kg.

Following PO dosing of lyophilized RX-5902 (83% GMP Lyophilized Cake:Group 3) at 65.9 mg/kg average dose, maximum plasma concentrations(average of 2027±359 ng/mL) were observed at 2 hours post dosing. Theaverage half-life was 9.70 hours. The average exposure based on the dosenormalized AUC_(Last) was 360±129 hr*kg*ng/mL/mg. The average oralbioavailability for RX-5902 (83% GMP Lyophilized Cake) was 18.2±6.53% atan average dose of 65.9 mg/kg.

Following PO dosing of high-energy milled lyophilized RX-5902 (83%High-Energy Milled Lyophilized Cake; Group 4) at 65.9 mg/kg averagedose, maximum plasma concentrations (average of 2613±692 ng/mL) wereobserved at 2 hours post dosing. The average half-life was 7.99 hours.The average exposure based on the dose normalized AUC_(Last) was456±45.9 hr*kg*ng/mL/mg. The average oral bioavailability for RX-5902(83% High-Energy Milled Lyophilized Cake) was 23.0±2.32% at an averagedose of 65.9 mg/kg.

Following PO dosing of Poloxamer spray dried RX-5902 [93% Spray DriedCake (SDM)+0.23% Poloxamer 407; Group 5] at 66.2 mg/kg, maximum plasmaconcentrations (average 1270±185 ng/mL) were observed between 2 and 4hours post dosing. The average half-life could not be determined;however, the half-life was 3.11 hours for Rat #588. The average exposurebased on the dose normalized AUC_(last) was 200±33.8 hr*kg*ng/mL/mg. Theaverage oral bioavailability for RX-5902 (93% Spray Dried Cake(SDM)+0.23% Poloxamer 407) was 10.1±1.71% at an average dose of 66.2mg/kg.

Following PO dosing of Poloxamer-free spray dried RX-5902 [93% SprayDried Cake (SDM); Group 6] at 65.6 mg/kg, maximum plasma concentrations(average of 1527±627 ng/mL) were observed between 2 and 4 hours postdosing. The average half-life could not be determined; however, thehalf-life was 6.89 hours for Rat #589. The average exposure based on thedose normalized AUC_(last) was 293±107 hr*kg*ng/mL/mg. The average oralbioavailability for RX-5902 (93% Spray Dried Cake (SDM) was 14.8±5.40%at an average dose of 65.6 mg/kg.

Oral dosing of high-energy milled lyophilized RX-5902 in Group 4 (83%High-Energy Milled Lyophilized Cake) had the highest oralbioavailability with an average of 23%. The overall rank order of oralbioavailability is Group 4 (83% High-Energy Milled LyophilizeCake) >Group 3 (83% GMP Lyophilized Cake)>Group 6 [93% Spray Dried Cake(SDM)]>Group 5 [93% Spray Dried Cake (SDM)+0.23% Poloxamer 407]>Group 1(Unmilled API in 0.36% Poloxamer 407 in ultrapure water). The oralbioavailability of differently nanoformulated materials of RX-5902 isshown in Table 9.

TABLE 9 Oral Bioavailability of Different Nanoformulated Materials ofRX-5902 Oral bio- availabil- Group Material ity (F) 1 Unmilled API(Preparation A) Dissolved in 7.62 ± 1.27% 0.36% Poloxamer 407 Solution(RX-5902 17.7 mg, 3.6 mg Poloxamer 407) PO Administration 2 Low-energyMilled Lyophilized Powder n/a (Preparation B) Dissolved in Water(RX-5902 10.6 mg, 2.1 mg Poloxamer 407) IV Administration 3 Low-energyMilled Lyophilized Powder 18.2 ± 6.53% (Preparation B) Dissolved inWater (RX-5902 17.5 mg, 3.6 mg Poloxamer 407) PO Administration 4High-energy Milled Lyophilized Powder 23.0 ± 2.32% (Preparation C)Dissolved in Water (RX-5902 17.4 mg, 3.6 mg Poloxamer 407) POAdministration 5 High-energy Milled Spray-dried Powder 10.1 ± 1.71%(Preparation D) Dissolved in 0.23% Poloxamer 407 Solution (RX-5902 17.5mg, 3.6 mg Poloxamer 407) PO Administration 6 High-energy MilledSpray-dried Powder 14.8 ± 5.40% (Preparation D) Dissolved in Water(RX-5902 17.4 mg, 1.3 mg of Poloxamer 407) PO Administration

For reference, the average oral bioavailability for RX-5902 (nanomilledsuspension) in fasted male and female dogs was 29.4% and 21.4%,respectively.

Example 18: RX-5902 API and Regioisomer Impurity

Summary:

A comparison of the analytical data for RX-5902 with the RRT 0.975Impurity was performed. The analytical data consisted of ¹H, ¹⁹F, ¹³CNMR, UV-Vis absorbance, and mass spectrometry (by LC-MS). All availableanalytical data strongly suggests that the RRT 0.975 Impurity is aregioisomer of RX-5902.

Background:

In some earlier batches of RX-5902, it was discovered that an unknownimpurity was not resolved completely from the main product peak asanalyzed using the HPLC method. A new HPLC method was developed whichwas able to resolve the unknown impurity (“RRT 0.975 Impurity”) from themain RX-5902 product peak.

In order to determine the identity of the RRT 0.975 Impurity, 15 gramsof a production batch of RX-5902 was separated using supercritical fluidchromatography (SFC). A RX-5902 fraction and RRT 0.975 Impurity fractionwere obtained. Using the new HPLC method with the Synergi HydroRPcolumn, the RX-5902 fraction was analyzed and found to be 97.7 area % ofRX-5902 with a major impurity of 1.5 area %. The RRT 0.975 Impurityfraction was also analyzed and found to be 79.0 area % of the RRT 0.975Impurity with 0.7 area % of RX-5902; there were also 4 impuritieseach >1 area %.

¹H, ¹⁹F, ¹³C NMR Data

¹H and ¹⁹F spectra were obtained on the RX-5902 fraction and the RRT0.975 Impurity fraction. The ¹³C NMR spectrum was obtained on only theRRT 0.975 Impurity fraction and compared with the ¹³C NMR spectrum of aprevious RX-5902 reference standard lot.

Based on an earlier synthetic step which likely generated aregioisomeric intermediate, it is speculated that the RRT 0.975 Impurityis also the regioisomer of RX-5902, whereby the fluorine atom is on theadjacent aromatic carbon. RX-5902 has the molecular formula C₂₂H₂₄FN₅O₄.

Proposed Structure for RRT 0.975 Impurity

FIG. 19 shows ¹H NMR spectrum of RX-5902; FIG. 20 shows ¹H NMR spectrumof RRT 0.975 Impurity; FIG. 21 shows overlay of ¹H NMR spectra of RRT0.975 Impurity (top) and RX-5902 (bottom); and FIG. 22 shows overlay of7.0-8.0 ppm region of ¹H NMR spectra of RRT 0.975 Impurity (top plot)and RX-5902 (bottom plot).

It can be seen that the two ¹H NMR spectra are very similar (especiallythe splitting patterns), with minor chemical shifts observed for thesignals in the 7.0-8.0 ppm region, and this observation stronglysuggests that the RRT 0.975 Impurity is a regioisomer of RX-5902.

FIG. 23 shows ¹³C NMR spectrum of RX-5902; FIG. 24 shows ¹³C NMRspectrum of RRT 0.975 Impurity; FIG. 25 shows overlay of ¹³C NMR spectraof RX-5902 (top plot) and RRT 0.975 Impurity (bottom plot); and FIG. 26shows of 108-150 ppm region of ¹³C NMR spectra of RX-5902 (top plot) andRRT 0.975 Impurity (bottom plot).

It can be seen that the two ¹³C NMR spectra are very similar, whichstrongly supports the possibility that the RRT 0.975 Impurity is aregioisomer of RX-5902.

FIG. 27 shows ¹⁹F NMR spectrum of RX-5902; FIG. 28 shows ¹⁹F NMRspectrum of RRT 0.975 Impurity.

As shown in FIGS. 27 and 28, the two ¹⁹F NMR spectra are quitedifferent. The ¹⁹F chemical shift is −114.5 ppm for RX-5902 while it is−112.0 ppm (appearing as a quartet) for the RRT 0.975 Impurity. Thisindicates that the fluorine atom is in a slightly different environmentand again strongly supports the possibility that the RRT 0.975 Impurityis a regioisomer of RX-5902.

UV-Vis Absorbance Data

UV-Vis absorbance data was gathered on a production batch of RX-5902separated using the same HPLC method. As shown in FIG. 29, the graphindicates that both RX-5902 (solid line) and the RRT 0.975 Impurity(dashed line) have very similar absorbance spectra. This again stronglysupports the possibility that the RRT 0.975 Impurity is a regioisomer ofRX-5902.

LC-MS Data

FIG. 30 shows LC-MS of 17.9 min peak corresponding to the main RX-5902product (the graph on the left overlaps of the three graphs from theright); FIG. 31 shows LC-MS of 17.2 min peak corresponding to the RRT0.975 Impurity (the graph on the left overlaps of the three graphs fromthe right); and FIG. 32 shows LC-MS data indicating that both RX-5902and the RRT 0.975 Impurity have the exact [M+Na]⁺ mass of 464, againsupporting the conclusion that the RRT 0.975 Impurity is a regioisomerof RX-5902.

Conclusion:

A comparison of the analytical data (consisting of ¹H, ¹⁹F, ¹³C NMR,UV-Vis absorbance, and mass spectrometry) strongly indicate that the RRT0.975 Impurity is a regioisomer of RX-5902.

Example 19: X-Ray Crystal Structure of RX-5902

Method of Analysis

The single crystal X-ray structure of RX-5902 was determined at 100 K inthe triclinic, space group P-1 using a crystal as grown. There is onefully ordered API molecule in the asymmetric unit. The final R1[I>2σ(I)]=4.77%. An XRPD pattern was calculated from the crystalstructure, which shows that the single crystal structure isrepresentative of the supplied material.

For XRPD analysis, a PANalytical (X'Pert³ Powder) X-ray powderdiffractometer and Si zero background holder were used. The parametersused are listed in Table 10.

TABLE 10 Parameters for XRPD test Parameter Value X-Ray wavelength Cu,kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426 Kα2/Kα1 intensity ratio: 0.50X-Ray tube setting 45 kV, 40 mA Divergence slit Automatic Scan modeContinuous Scan range (°2TH) 3°-40° Step size (°2TH) 0.16 Total time(min) 4 min

Example 19A XRPD of RX-5902 Nanoformulation

The XRPD of particles from a RX-5902 nanoformulation prepared accordingto the method of Example 16B was determined. As shown in FIG. 33,RX-5902 is crystalline. Detailed XRPD peak identification are found inTable 11.

TABLE 11 XRPD peak selection of RXN1490A-001-4 (B004194-12-A) Pos.Height FWHM Left d-spacing Rel. Int. [°2Th.] [cts] [°2Th.] [Å] [%]6.642046 348.056300 0.230256 13.30800 15.42 8.558004 2079.1550000.204672 10.33244 92.11 12.968890 320.995500 0.127920 6.82647 14.2214.346660 876.465800 0.102336 6.17385 38.83 14.753260 178.5746000.102336 6.00460 7.91 15.328680 765.607200 0.153504 5.78046 33.9215.574780 1790.413000 0.153504 5.68967 79.32 15.850830 1411.9450000.102336 5.59121 62.55 16.970760 1003.171000 0.281424 5.22467 44.4417.671800 539.593000 0.153504 5.01896 23.90 18.141790 1518.7270000.076752 4.88998 67.28 18.544620 513.062700 0.102336 4.78466 22.7319.727840 402.150200 0.153504 4.50028 17.82 20.506980 529.0903000.089544 4.33102 23.44 21.479630 1569.747000 0.051168 4.13705 69.5421.837050 709.123000 0.102336 4.07014 31.41 22.167590 269.3865000.127920 4.01019 11.93 22.923880 215.980500 0.204672 3.87957 9.5723.658390 1493.694000 0.115128 3.76076 66.17 24.417090 761.9805000.102336 3.64560 33.76 24.852430 2257.320000 0.191880 3.58272 100.0025.533150 377.178100 0.102336 3.48872 16.71 26.636410 222.1213000.153504 3.34668 9.84 27.474790 2110.269000 0.153504 3.24643 93.4928.174960 326.430600 0.179088 3.16733 14.46 28.796790 137.2651000.153504 3.10033 6.08 29.856940 54.482390 0.153504 2.99262 2.4130.860610 212.309100 0.153504 2.89754 9.41 31.366870 81.582460 0.1535042.85192 3.61 32.276590 111.028900 0.281424 2.77359 4.92 34.449570122.052000 0.153504 2.60345 5.41 35.664690 93.923380 0.204672 2.517494.16 36.904290 45.109600 0.153504 2.43572 2.00 37.463500 34.1544100.307008 2.40064 1.51 38.747700 19.893850 0.307008 2.32399 0.88

Example 19B XRPD of RX-5902 API

FIG. 34 shows XRPD pattern of RX-5902 API prepared according to themethod of Example 14.

Example 19B1—Solubility Assessment, Slow Cooling and Slow Evaporation

RX-5902 (ca. 2 mg) was weighed in a 1.5 ml clear glass vials. An aliquotof the corresponding solvent or solvent mixture was added at RT andsolubility was assessed after 10 min as shown in Table 12. Solutions andsuspensions were then placed into a shaking chamber at 50° C. for 10more minutes. If no dissolution was observed, another aliquot of solventwas added and samples were placed in the shaking chamber at 50° C. for10 more minutes. This procedure was repeated until dissolution wasachieved or a maximum of 400 vol was added.

TABLE 12 Solubility Assessment of RX-5902 Solubility Solubility Resultat RT at 50° C. after slow Result after Solvent (mg/ml) (mg/ml) coolingevaporation Methanol <100 ≥2.5 Solution Solution Ethanol <100 ≥2.5Solution Suspension, needles Acetone <100 ≥12.5 plate-like n/a crystalsMEK <100 ≥12.5 plate-like n/a crystals MIBK <100 ≥6.25 Suspension, n/aneedles Ethyl <100 ≥12.5 plate-like n/a Acetate crystals THF <100 ≥25Solution Dry white solid Acetonitrile <100 ≥12.5 plate-like n/a crystalsDMSO <100 ≥100 Solution Solution Isopropanol- <100 ≥2.5 SolutionSuspension, 10% water needles Legend: n/a, not applicable

Depending on the results obtained from the solubility assessment sampleswere treated as follows: (1) solutions obtained at RT were allowed toslowly evaporate at RT by piercing a needle on the vial cap, (2)solutions obtained at 50° C. were cooled at 0.25° C./min to 5° C.Solutions obtained after cooling were allowed to slowly evaporate at RT.Any suspensions obtained with crystals potentially suitable for SCXRDwere assessed by PLM microscopy and the most promising crystals wereused for SCXRD analyses.

Example 19B2—Single Crystal Structure Determination

Crystals of RX-5209 were crystallized from ethanol by slow evaporation(ca. 2 mg in 400 vol. (0.8 ml) of solvent). The crystals obtained wereof needle morphology. A crystal of sufficient size and quality foranalysis by single crystal X-ray diffraction was isolated withapproximate dimensions 0.650×0.080×0.070 mm. Optical micrographs of thecrystals as received and the single crystal used for the data collectionare shown in FIGS. 35A and 35B. Parameters and results of themeasurement are shown in Tables 13-21.

Crystallographic Tables

TABLE 13 Sample and crystal data. Crystallization solvents EthanolCrystallization method Slow evaporation Empirical formula C₂₂H₂₄FN₅O₄Formula weight 441.46 Temperature 100(2) K Wavelength 1.54178 Å Crystalsize 0.650 × 0.080 × 0.070 mm Crystal habit Colorless Needle Crystalsystem Triclinic Space group P-1 Unit cell dimensions a = 6.7439(3) Å α= 67.972(5) ° b = 11.4634(5) Å β = 86.247(4) ° c = 14.5456(8) Å γ =86.663(4) ° Volume 1039.48(9) Å3 Z 2 Density (calculated) 1.410 Mg/m³Absorption coefficient 0.880 mm⁻¹ F(000) 464

TABLE 14 Data collection and structure refinement. DiffractometerSuperNova, Dual, Cu at zero, Atlas Radiation source SuperNova (Cu) X-raySource, CuKα Data collection method omega scans Theta range for datacollection 9.018 to 74.481° Index ranges −6 ≤ h ≤ 8, −14 ≤ k ≤ 14, −18 ≤l ≤ 17 Reflections collected 9202 Independent reflections 4227 [R(int) =0.0459] Coverage of independent 99.4% reflections Variation in checkreflections n/a Absorption correction Semi-empirical from equivalentsMax. and min. transmission 1.00000 and 0.72908 Structure solutiontechnique Direct methods Structure solution program SHELXTL (Sheldrick,2013) Refinement technique Full-matrix least-squares on F² Refinementprogram SHELXTL (Sheldrick, 2013) Function minimized Σ w(F_(o) ² − F_(c)²)² Data/restraints/parameters 4227/0/296 Goodness-of-fit on F² 1.035Δ/σ_(max) 0.000 Final R indices 3240 data; I > 2σ(I) R1 = 0.0477, wR2 =0.1257 all data R1 = 0.0647, wR2 = 0.1397 Weighting scheme w = 1/[σ²(F_(o) ²) + (0.0750P)² + 0.1242P] where P = (F_(o) ² + 2F_(c) ²)/3Extinction coefficient n/a Largest diff. peak and hole 0.249 and −0.273eÅ−3

Refinement Summary:

Ordered Non-H atoms, XYZ Freely refining Ordered Non-H atoms, UAnisotropic H atoms (on carbon), XYZ Idealized positions riding onattached atoms H atoms (on carbon), U Appropriate multiple of U(eq) forbonded atom H atoms (on heteroatoms), XYZ Freely refining H atoms (onheteroatoms), U Isotropic Disordered atoms, OCC No disorder Disorderedatoms, XYZ No disorder Disordered atoms, U No disorder

TABLE 15 Atomic coordinates and equivalent isotropic atomic displacementparameters, (Å²). x/a y/b z/c U(eq) F1 0.33930(18) 0.50175(11)−0.12020(9) 0.0282(3) O1 0.23176(18) 0.04077(12) 0.47864(10) 0.0214(3)O2 −0.31711(18) 0.09001(12) 0.30184(10) 0.0207(3) O3 1.02089(19)0.70023(13) 0.38735(10) 0.0232(3) O4 0.6086(2) 0.87007(13) 0.10089(11)0.0278(3) C1 0.2064(3) 0.42287(17) −0.05502(14) 0.0220(4) N1 0.1173(2)0.26781(14) 0.21122(12) 0.0176(3) N2 −0.2182(2) 0.19787(14) 0.13777(12)0.0196(3) N3 −0.0078(2) 0.15134(14) 0.37225(12) 0.0179(3) N4 0.2561(2)0.25262(14) 0.40644(11) 0.0168(3) N5 0.4667(2) 0.47744(14) 0.36062(11)0.0166(3) C2 0.0448(3) 0.38960(18) −0.09454(14) 0.0222(4) C3 −0.0951(3)0.31417(18) −0.02920(14) 0.0224(4) C4 −0.0741(3) 0.27209(16) 0.07350(14)0.0189(4) C5 −0.1907(2) 0.16298(16) 0.23162(14) 0.0173(3) C6 −0.0189(2)0.19765(16) 0.27046(13) 0.0163(3) C7 0.0921(3) 0.30631(16) 0.11079(14)0.0179(3) C8 0.2363(3) 0.38315(17) 0.04389(14) 0.0210(4) C9 0.1691(2)0.14402(16) 0.42264(13) 0.0165(3) C10 0.1522(2) 0.37615(16) 0.37214(13)0.0172(3) C11 0.2900(2) 0.47736(16) 0.30681(13) 0.0172(3) C12 0.5727(2)0.35405(16) 0.38973(14) 0.0182(4) C13 0.4403(2) 0.24899(17) 0.45586(13)0.0176(3) C14 0.5861(2) 0.58318(16) 0.31906(13) 0.0165(3) C15 0.7508(2)0.59303(16) 0.37003(13) 0.0174(3) C16 0.8660(2) 0.69848(17) 0.33020(14)0.0177(3) C17 0.8272(3) 0.79508(17) 0.24001(14) 0.0191(4) C18 0.6636(3)0.78338(17) 0.19067(14) 0.0197(4) C19 0.5428(2) 0.68034(17) 0.22955(14)0.0191(4) C20 −0.4913(2) 0.05379(18) 0.26809(15) 0.0225(4) C21 1.1247(3)0.8144(2) 0.35825(17) 0.0308(5) C22 0.7554(3) 0.95587(19) 0.04173(15)0.0266(4)

U(eq) is defined as one third of the trace of the orthogonalized U_(ij)tensor.

TABLE 16 Selected bond length, (Å). F1—C1 1.364(2) O1—C9 1.227(2) O2—C51.341(2) O2—C20 1.442(2) O3—C16 1.382(2) O3—C21 1.427(2) O4—C18 1.370(2)O4—C22 1.434(2) C1—C8 1.361(3) C1—C2 1.400(3) N1—C6 1.302(2) N1—C71.378(2) N2—C4 1.386(2) N3—C6 1.378(2) N3—C9 1.423(2) N3—C9 1.423(2)N3—H3A 0.89(3) N4—C9 1.340(2) N4—C10 1.465(2) N4—C13 1.466(2) N5—C141.408(2) N5—C11 1.468(2) N5—C12 1.471(2) C2—C3 1.379(3) C3—C4 1.401(3)C4—C7 1.412(2) C5—C6 1.458(2) C7—C8 1.415(2) C10—C11 1.515(2) C12—C131.522(2) C14—C19 1.395(2) C14—C15 1.408(2) C15—C16 1.387(2) C16—C171.392(3) C17—C18 1.394(2) C18—C19 1.389(2)

TABLE 17 Selected bond angles, (°). C5—O2—C20 116.72(14) C16—O3—C21117.15(15) C18—O4—C22 117.38(15) C8—C1—F1 118.53(17) C8—C1—C2 124.02(17)F1—C1—C2 117.44(17) C6—N1—C7 116.91(15) C5—N2—C4 116.44(15) C6—N3—C9124.41(14) C6—N3—H3A 115.5(19) C9—N3—H3A 112.2(18) C9—N4—C10 124.08(14)C9—N4—C13 118.71(14) C10—N4—C13 113.84(14) C14—N5—C11 116.47(14)C14—N5—C12 115.86(13) C11—N5—C12 109.99(14) C3—C2—C1 117.99(17) C2—C3—C4120.56(17) N2—C4—C3 119.53(16) N2—C4—C7 120.53(17) C3—C4—C7 119.94(17)N2—C5—O2 122.67(15) N2—C5—C6 123.22(16) O2—C5—C6 114.10(16) N1—C6—N3122.18(16) N1—C6—C5 121.17(17) N3—C6—C5 116.65(15) N1—C7—C4 121.72(16)N1—C7—C8 118.68(16) C4—C7—C8 119.59(17) C1—C8—C7 117.87(17) O1—C9—N4123.90(16) O1—C9—N3 119.09(15) N4—C9—N3 117.01(15) N4—C10—C11 110.75(13)N5—C11—C10 109.96(14) N5—C12—C13 111.35(14) N4—C13—C12 109.79(14)C19—C14—C15 118.87(16) C19—C14—N5 121.69(15) C15—C14—N5 119.42(16)C16—C15—C14 119.45(16) O3—C16—C15 114.76(16) O3—C16—C17 122.84(15)C15—C16—C17 122.39(16) C16—C17—C18 117.23(16) O4—C18—C19 114.41(16)O4—C18—C17 123.79(16) C19—C18—C17 121.80(17) C18—C19—C14 120.23(16)

TABLE 18 Selected torsion angles, (°). C8—C1—C2—C3 1.5(3) F1—C1—C2—C3−177.32(16) C1—C2—C3—C4 −0.3(3) C5—N2—C4—C3 −179.49(16) C5—N2—C4—C70.0(2) C2—C3—C4—N2 179.06(16) C2—C3—C4—C7 −0.4(3) C4—N2—C5—O2−179.28(15) C4—N2—C5—C6 −0.2(2) C20—O2—C5—N2 −1.6(2) C20—O2—C5—C6179.27(14) C7—N1—C6—N3 178.67(15) C7—N1—C6—C5 −0.6(2) C9—N3—C6—N1−17.9(3) C9—N3—C6—C5 161.39(16) N2—C5—C6—N1 0.6(3) O2—C5—C6—N1179.71(15) N2—C5—C6—N3 −178.74(16) O2—C5—C6—N3 0.4(2) C6—N1—C7—C4 0.4(2)C6—N1—C7—C8 179.72(16) N2—C4—C7—N1 0.0(3) C3—C4—C7—N1 179.43(16)N2—C4—C7—C8 −179.39(16) C3—C4—C7—C8 0.1(3) F1—C1—C8—C7 176.99(15)C2—C1—C8—C7 −1.8(3) N1—C7—C8—C1 −178.41(16) C4—C7—C8—C1 1.0(3)C10—N4—C9—O1 −156.16(17) C13—N4—C9—O1 1.8(3) C10—N4—C9—N3 22.9(2)C13—N4—C9—N3 −179.15(15) C6—N3—C9—O1 −119.92(19) C6—N3—C9—N4 61.0(2)C9—N4—C10—C11 −147.08(16) C13—N4—C10—C11 53.99(19) C14—N5—C11—C10−166.06(14) C12—N5—C11—C10 59.55(18) N4—C10—C11—N5 −56.42(19)C14—N5—C12—C13 165.97(15) C11—N5—C12—C13 −59.34(19) C9—N4—C13—C12147.35(16) C10—N4—C13—C12 −52.49(19) N5—C12—C13—N4 54.67(19)C11—N5—C14—C19 −2.4(2) C12—N5—C14—C19 129.37(18) C11—N5—C14—C15176.08(15) C12—N5—C14—C15 −52.2(2) C19—C14—C15—C16 −0.1(2)N5—C14—C15—C16 −178.61(15) C21—O3—C16—C15 −170.41(17) C21—O3—C16—C179.2(3) C14—C15—C16—O3 178.42(15) C14—C15—C16—C17 −1.2(3) O3—C16—C17—C18−178.51(16) C15—C16—C17—C18 1.0(3) C22—O4—C18—C19 −160.61(17)C22—O4—C18—C17 19.0(3) C16—C17—C18—O4 −179.19(17) C16—C17—C18—C19 0.4(3)O4—C18—C19—C14 177.97(16) C17—C18—C19—C14 −1.6(3) C15—C14—C19—C18 1.5(3)N5—C14—C19—C18 179.92(16)

TABLE 19 Anisotropic atomic displacement parameters, (Å²). U11 U22 U33U23 U13 U12 F1 0.0346(6) 0.0267(6) 0.0213(6) −0.0068(5) 0.0082(5)−0.0096(5) O1 0.0171(6) 0.0164(6) 0.0245(7) −0.0004(5) 0.0012(5)−0.0043(5) O2 0.0168(6) 0.0221(7) 0.0226(7) −0.0070(5) 0.0007(5)−0.0078(5) O3 0.0222(6) 0.0215(7) 0.0257(7) −0.0071(5) −0.0044(5)−0.0081(5) O4 0.0278(7) 0.0216(7) 0.0261(7) 0.0020(5) −0.0061(6)−0.0076(5) C1 0.0249(9) 0.0164(9) 0.0239(9) −0.0074(7) 0.0054(7)−0.0037(7) N1 0.0157(6) 0.0163(7) 0.0204(7) −0.0062(6) 0.0000(6)−0.0028(5) N2 0.0199(7) 0.0162(7) 0.0225(8) −0.0066(6) −0.0026(6)−0.0020(6) N3 0.0148(6) 0.0173(7) 0.0201(7) −0.0044(6) −0.0006(6)−0.0060(5) N4 0.0131(6) 0.0156(7) 0.0204(7) −0.0050(5) −0.0016(5)−0.0017(5) N5 0.0130(6) 0.0147(7) 0.0215(7) −0.0054(6) −0.0022(5)−0.0027(5) C2 0.0285(9) 0.0196(9) 0.0184(8) −0.0069(7) −0.0030(7)0.0009(7) C3 0.0249(9) 0.0209(9) 0.0223(9) −0.0087(7) −0.0041(7)0.0007(7) C4 0.0192(8) 0.0147(8) 0.0235(9) −0.0078(7) −0.0001(7)−0.0015(6) C5 0.0163(8) 0.0120(8) 0.0244(9) −0.0072(6) −0.0024(7)−0.0023(6) C6 0.0158(7) 0.0125(8) 0.0203(8) −0.0055(6) −0.0007(6)−0.0022(6) C7 0.0188(8) 0.0140(8) 0.0203(8) −0.0060(6) 0.0000(7)−0.0015(6) C8 0.0201(8) 0.0195(9) 0.0242(9) −0.0091(7) 0.0022(7)−0.0042(7) C9 0.0126(7) 0.0188(8) 0.0169(8) −0.0054(6) 0.0023(6)−0.0033(6) C10 0.0138(7) 0.0157(8) 0.0216(8) −0.0059(7) −0.0007(6)−0.0027(6) C11 0.0144(7) 0.0156(8) 0.0207(8) −0.0053(6) −0.0027(6)−0.0008(6) C12 0.0159(7) 0.0147(8) 0.0226(9) −0.0049(6) −0.0012(7)−0.0023(6) C13 0.0140(7) 0.0183(8) 0.0196(8) −0.0056(6) −0.0024(6)−0.0016(6) C14 0.0131(7) 0.0166(8) 0.0211(8) −0.0087(7) 0.0012(6)−0.0017(6) C15 0.0158(7) 0.0165(8) 0.0194(8) −0.0063(6) 0.0013(6)−0.0024(6) C16 0.0146(7) 0.0191(8) 0.0226(9) −0.0111(7) −0.0013(6)−0.0015(6) C17 0.0170(7) 0.0170(8) 0.0225(9) −0.0065(7) 0.0030(7)−0.0053(6) C18 0.0188(8) 0.0182(9) 0.0210(9) −0.0058(7) −0.0014(7)−0.0007(6) C19 0.0151(7) 0.0193(9) 0.0233(9) −0.0082(7) −0.0012(7)−0.0020(6) C20 0.0128(8) 0.0237(9) 0.0328(10) −0.0120(8) −0.0004(7)−0.0064(6) C21 0.0323(10) 0.0259(10) 0.0338(11) −0.0078(8) −0.0072(8)−0.0145(8) C22 0.0292(9) 0.0207(9) 0.0250(9) −0.0024(7) 0.0002(8)−0.0064(7)

The anisotropic atomic displacement factor exponent takes the form:−2π²[h²a*² U₁₁+ . . . +2hka*b*U₁₂]

TABLE 20 Hydrogen atom coordinates and isotropic atomic displacementparameters, (Å²). x/a y/b z/c U H3A −0.088(4) 0.089(3) 0.406(2) 0.035(7)H2B 0.0318 0.4181 −0.1643 0.027 H3B −0.2067 0.2905 −0.0540 0.027 H8A0.3503 0.4063 0.0672 0.025 H10A 0.1021 0.3964 0.4301 0.021 H10B 0.03670.3739 0.3343 0.021 H11A 0.3308 0.4616 0.2458 0.021 H11B 0.2196 0.56070.2872 0.021 H12A 0.6918 0.3552 0.4256 0.022 H12B 0.6176 0.3375 0.32940.022 H13A 0.5112 0.1665 0.4697 0.021 H13B 0.4085 0.2593 0.5199 0.021H15A 0.7829 0.5280 0.4312 0.021 H17A 0.9087 0.8660 0.2132 0.023 H19A0.4304 0.6760 0.1950 0.023 H20A −0.5741 0.0030 0.3255 0.034 H20B −0.45080.0043 0.2273 0.034 H20C −0.5676 0.1294 0.2284 0.034 H21A 1.2215 0.80680.4078 0.046 H21B 1.0297 0.8843 0.3535 0.046 H21C 1.1941 0.8307 0.29360.046 H22A 0.7058 1.0031 −0.0244 0.040 H22B 0.8780 0.9086 0.0356 0.040H22C 0.7827 1.0146 0.0736 0.040

TABLE 21 Selected hydrogen bond information (Å and °). D-H . . . Ad(D-H) d(H . . . A) d(D . . . A) <(DHA) N3—H3A . . . O1#1 0.89(3)2.02(3) 2.8652(19) 160(3) #1−x, −y, −z + 1

Example 20: Analysis XRPD Data

An overlay comparing the XRPD patterns of RX-5902 nanoformulation(Example 18A (top)) and RX-5902 API (Example 18B (bottom)) is in FIG.36. Although these samples were analyzed at different times, the sametest method was used, which facilitates identification and comparison ofpeak positions. Review of the sample peak lists shows a good match onalmost all of the observed peaks. Variations in peak intensity and peaksplitting here are considered less significant and do not affect thematch. Note also that Poloxamer 407 reflections may contribute to thenanosuspension pattern. Both patterns are consistent with crystallinematerial; there is no obvious amorphous component. This datademonstrates that the nanosuspension crystalline structure is consistentwith RX-5902 API.

It will be apparent to those skilled in the art that specificembodiments of the disclosed subject matter may be directed to one ormore of the above- and below-indicated embodiments in any combination.While the invention has been disclosed in some detail by way ofillustration and example, it is apparent to those skilled in the artthat changes may be made and equivalents may be substituted withoutdeparting from the true spirit and scope of the invention. Therefore,the description and examples should not be construed as limiting thescope of the invention. All references, publications, patents, andpatent applications disclosed herein are hereby incorporated byreference in their entirety as if each had been individuallyincorporated.

The invention claimed is:
 1. A method of treating a tumor that expressesY593 phosphorylated p68, comprising administering to a human subject inneed thereof a solid, oral dosage form comprising a compound of formula(I)

or pharmaceutically acceptable salt thereof in an amount that inhibitsY593 phosphorylated p68 activity, wherein the solid, oral dosage formprovides an AUC_(0-t) (0-24 hours) of about 800-15,000 hr·ng/mL after asingle administration, wherein the solid, oral dosage form isadministered at a dosage of about 100-1,200 mg/day 1-7 days per week, upto about 2,800 mg/week.
 2. The method of claim 1, wherein the solid,oral dosage form is administered at a dosage of about 150-400 mg/day 3-7days per week.
 3. The method of claim 1, wherein the solid, oral dosageform is administered at a dosage of about 150-400 mg/day 5-7 days perweek.
 4. The method of claim 1, wherein the solid, oral dosage form is atablet or capsule.
 5. The method of claim 1, wherein the solid, oraldosage form provides an AUC_(0-t) (0-24 hours) of about 2,500-9,500hr·ng/mL after a single administration.
 6. The method of claim 1,wherein the solid, oral dosage form provides a C_(max) of about200-1,200 ng/mL after a single administration.
 7. The method of claim 1,further comprising administering radiation or an anti-tumor agent to thesubject.
 8. The method of claim 7, wherein the an anti-tumor agent isselected from the group consisting of antimetabolites, DNA-fragmentingagents, DNA-crosslinking agents, intercalating agents, protein synthesisinhibitors, topoisomerase I poisons, topoisomerase II poisons,microtubule-directed agents, kinase inhibitors, polyphenols, hormones,hormone antagonists, death receptor agonists, immune checkpointinhibitors, anti-programmed cell death 1 (PD-1) receptor antibodies andanti-programmed cell death ligand 1 (PD-L1) antibodies.
 9. The method ofclaim 1, further comprising administering to the subject a PD-L1antibody or PD-1 antibody.
 10. The method of claim 1, wherein theadministration inhibits β-catenin dependent ATPase activity of Y593phosphorylated p68.
 11. The method of claim 1, further comprising: (a)collecting a sample of the tumor from the subject before administeringthe solid, oral dosage form; and (b) determining whether the tumorexpresses Y593 phosphorylated p68.
 12. The method of claim 1, whereinthe tumor is selected from skin cancer, prostate cancer, colon cancer,ovarian cancer, breast cancer, lymphoma, and stomach cancer.